MN gene and protein

ABSTRACT

A new gene—MN—and proteins/polypeptides encoded therefrom are disclosed. Recombinant nucleic acid molecules for expressing MN proteins/polypeptides and recombinant proteins are provided. Expression of the MN gene is disclosed as being associated with tumorigenicity, and the invention concerns methods and compositions for detecting and/or quantitating MN antigen and/or MN-specific antibodies in vertebrate samples that are diagnostic/prognostic for neoplastic and pre-neoplastic disease. MN-specific antibodies are disclosed that can be used diagnostically/prognostically, therapeutically, for imaging, and/or for affinity purification of MN proteins/polypeptides. The invention still further concerns antisense nucleic acid sequences that can be used to inhibit MN gene expression.

This application is a continuation application of U.S. Ser. No.10/795,933 (filed Mar. 8, 2004) now abandoned which is a continuationapplication of U.S. Ser. No. 08/260,190 (filed Jun. 15, 1994) now U.S.Pat. No. 6,774,117 which is a continuation-in-part of U.S. Ser. No.08/177,093 (filed Dec. 30, 1993) which issued on Apr. 18, 2000 as U.S.Pat. No. 6,051,226, which is in turn a continuation-in-part of U.S. Ser.No. 07/964,589 (filed Oct. 21, 1992) which issued on Feb. 7, 1995 asU.S. Pat. No. 5,387,676. This application declares priority under 35 USC§120 from those U.S. applications, and also under 35 USC §119 from thenow pending Czechoslovakian patent application PV-709-92 (filed Mar. 11,1992).

FIELD OF THE INVENTION

The present invention is in the general area of medical genetics and inthe fields of biochemical engineering and immunochemistry. Morespecifically, it relates to the identification of a new gene—the MNgene—a cellular gene coding for the MN protein. The inventors hereoffound MN proteins to be associated with tumorigenicity. Evidenceindicates that the MN protein appears to represent a potentially noveltype of oncoprotein. Identification of MN antigen as well as antibodiesspecific therefor in patient samples provides the basis fordiagnostic/prognostic assays for cancer.

BACKGROUND OF THE INVENTION

A novel quasi-viral agent having rather unusual properties was detectedby its capacity to complement mutants of vesicular stomatitis virus(VSV) with heat-labile surface G protein in HeLa cells (cell linederived from human cervical adenocarcinoma), which had been cocultivatedwith human breast carcinoma cells. [Zavada et al., Nature New Biol.,240: 124 (1972); Zavada et al., J. Gen. Virol., 24: 327 (1974); Zavada,J., Arch. Virol., 50: 1 (1976); Zavada, J., J. Gen. Virol., 63: 15-24(1982); Zavada and Zavadova, Arch. Virol., 118: 189 (1991).] The quasiviral agent was called MaTu as it was presumably derived from a humanmammary tumor.

There was significant medical interest in studying and characterizingMaTu as it appeared to be an entirely new type of molecular parasite ofliving cells, and possibly originated from a human tumor. Describedherein is the elucidation of the biological and molecular nature of MaTuwhich resulted in the discovery of the MN gene and protein. MaTu wasfound by the inventors to be a two-component system, having an exogenoustransmissible component, MX, and an endogenous cellular component, MN.As described herein, the MN component was found to be a cellular gene,showing only very little homology with known DNA sequences. The MN genewas found to be present in the chromosomal DNA of all vertebratestested, and its expression was found to be strongly correlated withtumorigenicity.

The exogenous MaTu-MX transmissible agent was identified as lymphocyticchoriomeningitis virus (LCMV) which persistently infects HeLa cells. Theinventors discovered that the MN expression in HeLa cells is positivelyregulated by cell density, and also its expression level is increased bypersistent infection with LCMV.

Research results provided herein show that cells transfected with MNcDNA undergo changes indicative of malignant transformation. Furtherresearch findings described herein indicate that the disruption of cellcycle control is one of the mechanisms by which MN may contribute to thecomplex process of tumor development.

Described herein is the cloning and sequencing of the MN gene and therecombinant production of MN proteins. Also described are antibodiesprepared against MN proteins/polypeptides. MN proteins/polypeptides canbe used in serological assays according to this invention to detectMN-specific antibodies. Further, MN proteins/polypeptides and/orantibodies reactive with MN antigen can be used in immunoassaysaccording to this invention to detect and/or quantitate MN antigen. Suchassays may be diagnostic and/or prognostic for neoplastic/pre-neoplasticdisease.

SUMMARY OF THE INVENTION

Herein disclosed is the MN gene, a cellular gene which is the endogenouscomponent of the MaTu agent. cDNA sequences for the MN gene are shown inFIGS. 1A-B [SEQ. ID. NO.: 1] and FIG. 15 [SEQ. ID. NO.: 5]. FIG. 25provides the sequence of a MN genomic clone containing a promoter region[SEQ. ID. NO.: 23].

This invention is directed to said MN gene, fragments thereof and therelated cDNA which are useful, for example, as follows: 1) to produce MNproteins/polypeptides by biochemical engineering; 2) to prepare nucleicacid probes to test for the presence of the MN gene in cells of asubject; 3) to prepare appropriate polymerase chain reaction (PCR)primers for use, for example, in PCR-based assays or to produce nucleicacid probes; 4) to identify MN proteins and polypeptides as well ashomologs or near homologs thereto; 5) to identify various mRNAstranscribed from MN genes in various tissues and cell lines, preferablyhuman; and 6) to identify mutations in MN genes. The invention furtherconcerns purified and isolated DNA molecules comprising the MN gene orfragments thereof, or the related cDNA or fragments thereof.

Thus, this invention in one aspect concerns isolated nucleic acidsequences that encode MN proteins or polypeptides wherein the nucleotidesequences for said nucleic acids are selected from the group consistingof:

(a) SEQ. ID. NO.: 1;

(b) SEQ. ID. NO.: 5;

(c) nucleotide sequences that hybridize under stringent conditions toSEQ. ID. NO.: 1 or to its complement;

(d) nucleotide sequences that hybridize under stringent conditions toSEQ. ID. NO.: 5 or to its complement; and

(e) nucleotide sequences that differ from SEQ. ID. NO.: 1 or SEQ. IDNO.: 5, or from the nucleotide sequences of (c) and (d) in codonsequence because of the degeneracy of the genetic code, that is,sequences that are degenerate variants of those sequences. Further, suchnucleic acid sequences are selected from nucleotide sequences that butfor the degeneracy of the genetic code would hybridize to either SEQ.ID. NO.: 1 or SEQ. ID. NO.: 5 under stringent hybridization conditions.

Further, such isolated nucleic acids that encode MN proteins orpolypeptides can also include the MN nucleic acid of the genomic cloneshown in FIG. 25 a-b, that is, SEQ. ID. NO.: 23, as well as sequencesthat hybridize to it or its complement under stringent conditions, orwould hybridize to SEQ. ID. NO.: 23 or its complement under suchconditions, but for the degeneracy of the genetic code.

Further, this invention concerns nucleic acid probes which are fragmentsof the isolated nucleic acids that encode MN proteins or polypeptides asdescribed above. Preferably said nucleic acid probes are comprised of atleast 50 nucleotides, more preferably at least 100 nucleotides, andstill more preferably at least 150 nucleotides.

Still further, this invention is directed to isolated nucleic acidsselected from the group consisting of:

(a) a nucleic acid having the nucleotide sequence shown in FIG. 25 [SEQ.ID. NO.: 23] and its complement;

(b) nucleic acids that hybridize under standard stringent hybridizationconditions to the nucleic acid of (a) or to its complement; and

(c) nucleic acids that differ from the nucleic acids of (a) and (b) incodon sequence because of the degeneracy of the genetic code. Theinvention also concerns nucleic acids that but for the degeneracy of thegenetic code would hybridize to the nucleic acid of (a) or to itscomplement under standard stringent hybridization conditions. Thenucleic acids of (b) and (c) that hybridize to the coding region of SEQ.ID. NO.: 23 preferably have a length of at least 50 nucleotides, whereasthe nucleic acids of (b) and (c) that hybridize partially or wholly tothe non-coding region of SEQ. ID. NO.: 23 or its complement are thosethat function as nucleic acid probes to identify MN nucleic acidsequences. Conventional technology can be used to determine whether thenucleic acids of (b) and (c) or of fragments of SEQ. ID. NO.: 23 areuseful to identify MN nucleic acid sequences, for example, as outlinedin Benton and Davis, Science, 196: 180 (1977) and Fuscoe et al.Genomics, 5: 100 (1989). In general, the nucleic acids of (b) and (c)are preferably at least 50 nucleotides, more preferably at least 100nucleotides, and still more preferably at least 150 nucleotides.

Test kits of this invention can comprise the nucleic acid probes of theinvention which are useful diagnostically/prognostically for neoplasticand/or pre-neoplastic disease. Preferred test kits comprise means fordetecting or measuring the hybridization of said probes to the MN geneor to the mRNA product of the MN gene, such as a visualizing means.

Fragments of the isolated nucleic acids of the invention, can also beused as PCR primers to amplify segments of MN genes, and may be usefulin identifying mutations in MN genes. Typically, said PCR primers areoligonucleotides, preferably at least 16 nucleotides, but they may beconsiderably longer. Exemplary primers may be from about 16 nucleotidesto about 50 nucleotides, preferably from about 19 nucleotides to about45 nucleotides.

This invention also concerns nucleic acids which encode MN proteins orpolypeptides that are specifically bound by monoclonal antibodiesdesignated M75 that are produced by the hybridoma VU-M75 deposited atthe American Type Culture Collection (ATCC) 10801 University Blvd.,Manassas, Va. 20110-2209 (USA) under ATCC No. HB 11128, and/or bymonoclonal antibodies designated MN12 produced by the hybridoma MN12.2.2 deposited at the ATCC under ATCC No. HB 11647.

The invention further concerns the discovery of a hitherto unknownprotein—MN, encoded by the MN gene. The expression of MN proteins isinducible by growing cells in dense cultures, and such expression wasdiscovered to be associated with tumorigenic cells.

MN proteins were found to be produced by some human tumor cell lines invitro, for example, by HeLa (cervical carcinoma), T24 (bladdercarcinoma) and T47D (mammary carcinoma) and SK-MeI 1477 (melanoma) celllines, by tumorigenic hybrid cells and by cells of some human cancers invivo, for example, by cells of uterine cervical, ovarian and endometrialcarcinomas as well as cells of some benign neoplasias such as mammarypapillomas. MN proteins were not found in non-tumorigenic hybrid cells,and are generally not found in the cells of normal tissues, althoughthey have been found in a few normal tissues, most notably andabundantly in normal stomach tissues. MN antigen was found byimmunohistochemical staining to be prevalent in tumor cells and to bepresent sometimes in morphologically normal appearing areas of tissuespecimens exhibiting dysplasia and/or malignancy. Thus, the MN gene isstrongly correlated with tumorigenesis and is considered to be aputative oncogene.

In HeLa and in tumorigenic HeLa×fibroblast hybrid (H/F-T) cells, MNprotein is manifested as a “twin” protein p54/58N; it is glycosylatedand forms disulfide-linked oligomers. As determined by electrophoresisupon reducing gels, MN proteins have molecular weights in the range offrom about 40 kd to about 70 kd, preferably from about 45 kd to about 65kd, more preferably from about 48 kd to about 58 kd. Upon non-reducinggels, MN proteins in the form of oligomers have molecular weights in therange of from about 145 kd to about 160 kd, preferably from about 150 toabout 155 kd, still more preferably from about 152 to about 154 kd. Thepredicted amino acid sequences for preferred MN proteins of thisinvention are shown in FIG. 1A-1B [SEQ. ID. NO. 2] and in FIG. 15 [SEQ.ID. NO.: 6].

The discovery of the MN gene and protein and thus, of substantiallycomplementary MN genes and proteins encoded thereby, led to the findingthat the expression of MN proteins was associated with tumorigenicity.That finding resulted in the creation of methods that arediagnostic/prognostic for cancer and precancerous conditions. Methodsand compositions are provided for identifying the onset and presence ofneoplastic disease by detecting and/or quantitating MN antigen inpatient samples, including tissue sections and smears, cell and tissueextracts from vertebrates, preferably mammals and more preferablyhumans. Such MN antigen may also be found in body fluids.

MN proteins and genes are of use in research concerning the molecularmechanisms of oncogenesis, in cancer diagnostics/prognostics, and may beof use in cancer immunotherapy. The present invention is useful fordetecting a wide variety of neoplastic and/or pre-neoplastic diseases.Exemplary neoplastic diseases include carcinomas, such as mammary,bladder, ovarian, uterine, cervical, endometrial, squamous cell andadenosquamous carcinomas; and head and neck cancers; mesodermal tumors,such as neuroblastomas and retinoblastomas; sarcomas, such asosteosarcomas and Ewing's sarcoma; and melanomas. Of particular interestare head and neck cancers, gynecologic cancers including ovarian,cervical, vaginal, endometrial and vulval cancers; gastrointestinalcancer, such as, stomach, colon and esophageal cancers; urinary tractcancer, such as, bladder and kidney cancers; skin cancer; liver cancer;prostate cancer; lung cancer; and breast cancer. Of still furtherparticular interest are gynecologic cancers; breast cancer; urinarytract cancers, especially bladder cancer; lung cancer; and liver cancer.Even further of particular interest are gynecologic cancers and breastcancer. Gynecologic cancers of particular interest are carcinomas of theuterine cervix, endometrium and ovaries; more particularly suchgynecologic cancers include cervical squamous cell carcinomas,adenosquamous carcinomas, adenocarcinomas as well as gynecologicprecancerous conditions, such as metaplastic cervical tissues andcondylomas.

The invention further relates to the biochemical engineering of the MNgene, fragments thereof or related cDNA. For example, said gene or afragment thereof or related cDNA can be inserted into a suitableexpression vector; host cells can be transformed with such an expressionvector; and an MN protein/polypeptide, preferably an MN protein, isexpressed therein. Such a recombinant protein or polypeptide can beglycosylated or nonglycosylated, preferably glycosylated, and can bepurified to substantial purity. The invention further concerns MNproteins/polypeptides which are synthetically or otherwise biologicallyprepared.

Said MN proteins/polypeptides can be used in assays to detect MN antigenin patient samples and in serological assays to test for MN-specificantibodies. MN proteins/polypeptides of this invention are serologicallyactive, immunogenic and/or antigenic. They can further be used asimmunogens to produce MN-specific antibodies, polyclonal and/ormonoclonal, as well as an immune T-cell response.

The invention further is directed to MN-specific antibodies, which canbe used diagnostically/prognostically and may be used therapeutically.Preferred according to this invention are MN-specific antibodiesreactive with the epitopes represented respectively by the amino acidsequences of the MN protein shown in FIG. 15 as follows: from AA 62 toAA 67 [SEQ. ID. NO.: 10]; from AA 55 to AA 60 [SEQ. ID. NO.: 11]; fromAA 127 to AA 147 [SEQ. ID. NO.: 12]; from AA 36 to AA 51 [SEQ. ID. NO.:13]; from AA 69 to AA 83 [SEQ. ID. NO.: 14]; from AA 279 to AA 291 [SEQ.ID. NO.: 15]; and from AA 450 to AA 462 [SEQ. ID. NO.: 16]. Morepreferred are antibodies reactive with epitopes represented by SEQ. ID.NOS.: 10, 11 and 12. Still more preferred are antibodies reactive withthe epitopes represented by SEQ. ID NOS: 10 and 11, as for example,respectively Mabs M75 and MN12. Most preferred are monoclonal antibodiesreactive with the epitope represented by SEQ. ID. NO.: 10.

Also preferred according to this invention are antibodies preparedagainst recombinantly produced MN proteins as, for example, GEX-3X-MNand MN 20-19. Also preferred are MN-specific antibodies prepared againstglycosylated MN proteins, such as, MN 20-19 expressed in baculovirusinfected Sf9 cells.

A hybridoma that produces a representative MN-specific antibody, themonoclonal antibody M75 (Mab M75), was deposited at the ATCC underNumber HB 11128 as indicated above. The M75 antibody was used todiscover and identify the MN protein and can be used to identify readilyMN antigen in Western blots, in radioimmunoassays andimmunohistochemically, for example, in tissue samples that are fresh,frozen, or formalin-, alcohol-, acetone- or otherwise fixed and/orparaffin-embedded and deparaffinized. Another representative MN-specificantibody, Mab MN12, is secreted by the hybridoma MN 12.2.2, which wasdeposited at the ATCC under the designation HB 11647.

MN-specific antibodies can be used, for example, in laboratorydiagnostics, using immunofluorescence microscopy or immunohistochemicalstaining; as a component in immunoassays for detecting and/orquantitating MN antigen in, for example, clinical samples; as probes forimmunoblotting to detect MN antigen; in immunoelectron microscopy withcolloid gold beads for localization of MN proteins and/or polypeptidesin cells; and in genetic engineering for cloning the MN gene orfragments thereof, or related cDNA. Such MN-specific antibodies can beused as components of diagnostic/prognostic kits, for example, for invitro use on histological sections; such antibodies can also and usedfor in vivo diagnostics/prognostics, for example, such antibodies can belabeled appropriately, as with a suitable radioactive isotope, and usedin vivo to locate metastases by scintigraphy. Further such antibodiesmay be used in vivo therapeutically to treat cancer patients with orwithout toxic and/or cytostatic agents attached thereto. Further, suchantibodies can be used in vivo to detect the presence of neoplasticand/or pre-neoplastic disease. Still further, such antibodies can beused to affinity purify MN proteins and polypeptides.

This invention also concerns recombinant DNA molecules comprising a DNAsequence that encodes for an MN protein or polypeptide, and alsorecombinant DNA molecules that encode not only for an MN protein orpolypeptide but also for an amino acid sequence of a non-MN protein orpolypeptide. Said non-MN protein or polypeptide may preferably benonimmunogenic to humans and not typically reactive to antibodies inhuman body fluids. Examples of such a DNA sequence is the alpha-peptidecoding region of beta-galactosidase and a sequence coding forglutathione S-transferase or a fragment thereof. However, in someinstances, a non-MN protein or polypeptide that is serologically active,immunogenic and/or antigenic may be preferred as a fusion partner to aMN antigen. Further, claimed herein are such recombinant fusionproteins/polypeptides which are substantially pure and non-naturallyoccurring. An exemplary fusion protein of this invention is GEX-3X-MN.

This invention also concerns methods of treating neoplastic diseaseand/or pre-neoplastic disease comprising inhibiting the expression of MNgenes by administering antisense nucleic acid sequences that aresubstantially complementary to mRNA transcribed from MN genes. Saidantisense nucleic acid sequences are those that hybridize to such mRNAunder stringent hybridization conditions. Preferred are antisensenucleic acid sequences that are substantially complementary to sequencesat the 5′ end of the MN cDNA sequences shown in FIG. 1A-1B and/or inFIG. 15. Preferably said antisense nucleic acid sequences areoligonucleotides.

This invention also concerns vaccines comprising an immunogenic amountof one or more substantially pure MN proteins and/or polypeptidesdispersed in a physiologically acceptable, nontoxic vehicle, whichamount is effective to immunize a vertebrate, preferably a mammal, morepreferably a human, against a neoplastic disease associated with theexpression of MN proteins. Said proteins can be recombinantly,synthetically or otherwise biologically produced. Recombinent MNproteins include GEX-3X-MN and MN 20-19. A particular use of saidvaccine would be to prevent recidivism and/or metastasis. For example,it could be administered to a patient who has had an MN-carrying tumorsurgically removed, to prevent recurrence of the tumor.

The immunoassays of this invention can be embodied in test kits whichcomprise MN proteins/polypeptides and/or MN-specific antibodies. Suchtest kits can be in solid phase formats, but are not limited thereto,and can also be in liquid phase format, and can be based onimmunohistochemical assays, ELISAs, particle assays, radiometric orfluorometric assays either unamplified or amplified, using, for example,avidin/biotin technology.

Abbreviations

The following abbreviations are used herein;

AA amino acid ATCC American Type Culture Collection bp base pairs BSAbovine serum albumin BRL Bethesda Research Laboratories CA carbonicanhydrase Ci curie cm centimeter cpm counts per minute C-terminuscarboxyl-terminus ° C. degrees centigrade DAB diaminobenzidine dH₂0deionized water DMEM Dulbecco modified Eagle medium DTT dithiothreitolEDTA ethylenediaminetetracetate EIA enzyme immunoassay ELISAenzyme-linked immunosorbent assay EtOH ethanol F fibroblasts FCS fetalcalf serum FIBR fibroblasts FITC fluorescein isothiocyanate GEX-3X-MNfusion protein MN glutathione S-transferase H HeLa cells H₂0₂ hydrogenperoxide HCA Hydrophobic Cluster Analysis HEF human embryo fibroblastsHeLa K standard type of HeLa cells HeLa S Stanbridge's mutant HeLaD98/AH.2 H/F-T hybrid HeLa fibroblast cells that are tumorigenic;derived from HeLa D98/AH.2 H/F-N hybrid HeLa fibroblast cells that arenontumorigenic; derived from HeLa D98/AH.2 HGPRT⁻ hypoxanthine guaninephosphoribosyl transferase-deficient HLH helix-loop-helix HRPhorseradish peroxidase IPTG isopropyl-beta-D-thiogalacto-pyranoside kbkilobase kbp kilobase pairs kd kilodaltons KPL Kirkegaard & PerryLaboratories, Inc. LCMV lymphocytic choriomeningitis virus LTR longterminal repeat M molar mA milliampere MAb monoclonal antibody MEmercaptoethanol MEM minimal essential medium min. minute(s) mg milligramml milliliter mM millimolar MMC mitomycin C MTV mammary tumor virus Nnormal concentration ng nanogram NGS normal goat serum N-terminusamino-terminus ODN oligodeoxynucleotide PAGE polyacrylamide gelelectrophoresis PBS phosphate buffered saline PCR polymerase chainreaction PEST combination of one-letter abbreviations for proline,glutamic acid, serine, threonine pI isoelectric point RIPradioimmunoprecipitation RIPA radioimmunoprecipitation assay SAC proteinA-Staphylococcus aureus cells SDRE serum dose response element SDSsodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate-polyacrylamidegel electrophoresis SP-RIA solid-phase radioimmunoassay SSPE NaCl(0.18M), sodium phosphate (0.01M), EDTA (0.001M) TBE Tris-borate/EDTAelectrophoresis buffer TCA trichloroacetic acid TC media tissue culturemedia TMB tetramethylbenzidine Tris tris (hydroxymethyl) aminomethaneμCi microcurie μg microgram μl microliter μM micromolar VSV vesicularstomatitis virus X-MLV xenotropic murine leukemia virus

Cell Lines

The following cell lines were used in the experiments herein described:

HeLa K standard type of HeLa cells; aneuploid, epithelial-like cell lineisolated from a human cervical adenocarcinoma [Gey et al., Cancer Res.,12: 264 (1952); Jones et al., Obstet. Gynecol., 38: 945-949 (1971)]obtained from Professor B. Korych, [Institute of Medical Microbiologyand Immunology, Charles University; Prague, Czech Republic] HeLaD98/AH.2 Mutant HeLa clone that is hypoxanthine (also HeLa S) guaninephosphoribosyl transferase- deficient (HGPRT⁻) kindly provided by EricJ. Stanbridge [Department of Microbiology, College of Medicine,University of California, Irvine, CA (USA)] and reported in Stanbridgeet al., Science, 215: 252-259 (15 Jan. 1982); parent of hybrid cellsH/F-N and H/F-T, also obtained from E. J. Stanbridge. NIH-3T3 murinefibroblast cell line reported in Aaronson, Science, 237: 178 (1987).T47D cell line derived from a human mammary carcinoma [Keydar et al.,Eur. J. Cancer, 15: 659-670 (1979)]; kindly provided by J. Keydar[Haddasah Medical School; Jerusalem, Israel] T24 cell line from urinarybladder carcinoma [Bubenik et al., Int. J. Cancer, 11: 765-773 (1973)]kindly provided by J. Bubenik [Institute of Molecular Genetics,Czechoslovak Academy of Sciences; Prague, Czech Republic] HMB2 cell linefrom melanoma [Svec et al., Neoplasma, 35: 665-681 (1988)] HEF humanembryo fibroblasts [Zavada et al., Nature New Biology, 240: 124-125(1972)] SIRC cell line from rabbit cornea (control and X-MLV- infected)[Zavada et al., Virology, 82: 221-231 (1977)] Vero cells African greenmonkey cell line [Zavada et al. (1977)] myeloma cell myeloma cell lineused as a fusion parent line NS-0 in production of monoclonal antibodies[Galfre and Milstein, Methods Enzymol., 73: 3-46 (1981)] SK-Mel 1477human melanoma cell line kindly provided by K. E. Hellstrom [Division ofTumor Immunology, Fred Hutchins Cancer Research Center; Seattle,Washington (USA)] XC cells derived from a rat rhabdomyosarcoma inducedwith Rous sarcoma virus-induced rat sarcoma [Svoboda, J., Natl. CancerCenter Institute Monograph No. 17, IN: “International Conference onAvian Tumor Viruses” (J. W. Beard ed.), pp. 277-298 (1964)], kindlyprovided by Jan Svoboda [Institute of Molecular Genetics, CzechoslovakAcademy of Sciences; Prague, Czech Republic]; and Rat 2-Tk⁻ a thymidinekinase deficient cell line, kindly provided by L. Kutinova [Institute ofSera and Vaccines; Prague, Czech Republic] CGL1 H/F-N hybrid cells (HeLaD98/AH.2 derivative) CGL2 H/F-N hybrid cells (HeLa D98/AH.2 derivative)CGL3 H/F-T hybrid cells (HeLa D98/AH.2 derivative) CGL4 H/F-T hybridcells (HeLa D98/Ah.2 derivative)

Nucleotide and Amino Acid Sequence Symbols

The following symbols are used to represent nucleotides herein:

Base Symbol Meaning A adenine C cytosine G guanine T thymine U uracil Iinosine M A or C R A or G W A or T/U S C or G Y C or T/U K G or T/U V Aor C or G H A or C or T/U D A or G or T/U B C or G or T/U N/X A or C orG or T/U

There are twenty main amino acids, each of which is specified by adifferent arrangement of three adjacent nucleotides (triplet code orcodon), and which are linked together in a specific order to form acharacteristic protein. A three-letter or one-letter convention is usedherein to identify said amino acids, as, for example, in FIGS. 1A-B andFIG. 15, respectively, as follows:

3 Ltr. 1 Ltr. Amino acid name Abbrev. Abbrev. Alanine Ala A Arginine ArgR Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid GluE Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV Unknown or other X

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1B provides the nucleotide sequence for a MN cDNA [SEQ. ID. NO.:1] clone isolated as described herein and the predicted amino acidsequence [SEQ. ID. NO.: 2] encoded by the cDNA. That sequence data hasbeen sent to the EMBL Data Library in Heidelberg, Germany and isavailable under Accession No. X66839.

FIG. 2 provides SDS-PAGE and immunoblotting analyses of recombinant MNprotein expressed from a pGEX-3X bacterial expression vector. Twoparallel samples of purified recombinant MN protein (twenty μg in eachsample) were separated by SDS-PAGE on a 10% gel. One sample (A in FIG.2) was stained with Coomassie brilliant blue; whereas the other sample(B) was blotted onto a Hybond C membrane [Amersham; Aylesbury, Bucks,England]. The blot was developed by autoradiography with ¹²⁵I-labeledMab M75.

FIG. 3 illustrates inhibition of p54/58 expression by antisenseoligodeoxynucleotides (ODNs). HeLa cells cultured in overcrowdedconditions were incubated with (A) 29-mer ODNI [SEQ. ID. NO.: 3]; (B)19-mer ODN2 [SEQ. ID. NO.: 4]; (C) both ODNI and ODN2; and (D) withoutODNS. Example 10 provides details of the procedures used.

FIG. 4 shows the results of Northern blotting of MN mRNA in human celllines. Total RNA was prepared from the following cell lines: HeLa cellsgrowing in dense (A) and sparse (B) culture; (C) H/F-N; (D) and (E)H/F-T; and (F) human embryo fibroblasts. Example 11 details theprocedure and results.

FIG. 5 illustrates the detection of the MN gene in genomic DNAs bySouthern blotting. Chromosomal DNA digested by PstI was as follows: (A)chicken; (B) bat; (C) rat; (D) mouse; (E) feline; (F) pig; (G) sheep;(H) bovine; (I) monkey; and (J) human HeLa cells. The procedures usedare detailed in Example 12.

FIG. 6 graphically illustrates the expression of MN- and MX-specificproteins in human fibroblasts (F), in HeLa cells (H) and in H/F-N andH/F-T hybrid cells and contrasts the expression in MX-infected andMX-uninfected cells. Example 5 details the procedures and results.

FIG. 7 (discussed in Example 5) provides immunoblots of MN proteins infibroblasts (FIBR) and in HeLa K, HeLa S, H/F-N and H/F-T hybrid cells.

FIG. 8 (discussed in Example 6) shows immunoblots of MN proteins in cellculture extracts prepared from the following: (A) MX-infected HeLacells; (B) human fibroblasts; (C) T24; (D) T47D; (E) SK-Mel 1477; and(F) HeLa cells uninfected with MX. The symbols +ME and O ME indicatethat the proteins were separated by PAGE after heating in a samplebuffer, with and without 3% mercaptoethanol (ME), respectively.

FIG. 9 (discussed in Example 6) provides immunoblots of MN proteins fromhuman tissue extracts. The extracts were prepared from the following:(A) MX-infected HeLa cells; (B) full-term placenta; (C) corpus uteri;(D, M) adenocarcinoma endometrii; (E, N) carcinoma ovarii; (F, G)trophoblasts; (H) normal ovary; (I) myoma uteri; (J) mammary papilloma;(K) normal mammary gland; (L) hyperplastic endometrium; (O) cervicalcarcinoma; and (P) melanoma.

FIG. 10 (discussed in Example 7) provides immunoblots of MN proteinsfrom (A) MX-infected HeLa cells and from (B) Rat2-TK⁻ cells. (+ME and 0ME have the same meanings as explained in the legend to FIG. 8.)

FIG. 11 (discussed in Example 8) graphically illustrates the resultsfrom radioimmunoprecipitation experiments with ¹²⁵I-GEX-3X-MN proteinand different antibodies. The radioactive protein (15×10³ cpm/tube) wasprecipitated with ascitic fluid or sera and SAC as follows: (A) asciteswith MAb M75; (B) rabbit anti-MaTu serum; (C) normal rabbit serum; (D)human serum L8; (E) human serum KH; and (F) human serum M7.

FIG. 12 (discussed in Example 8) shows the results fromradioimmunoassays for MN antigen. Ascitic fluid (dilution precipitating50% radioactivity) was allowed to react for 2 hours with (A) “cold”(unlabeled) protein GEX-3X-MN, or with extracts from cells as follows:(B) HeLa+MX; (C) Rat-2Tk⁻; (D) HeLa; (E) rat XC; (F) T24; and (G) HEF.Subsequently ¹²⁵I-labeled GEX-3X-MN protein (25×10³ cpm/tube) was addedand incubated for an additional 2 hours. Finally, the radioactivity toMAb M75 was adsorbed to SAC and measured.

FIG. 13 (discussed in Example 9) provides results of immunoelectron andscanning microscopy of MX-uninfected (control) and MX-infected HeLacells. Panels A-D show ultrathin sections of cells stained with MAb M75and immunogold; Panels E and F are scanning electron micrographs ofcells wherein no immunogold was used. Panels E and F both show aterminal phase of cell division. Panels A and E are of control HeLacells; panels B, C, D and F are of MX-infected HeLa cells. The cellsshown in Panels A, B and C were fixed and treated with M75 andimmunogold before they were embedded and sectioned. Such a procedureallows for immunogold decoration only of cell surface antigens. Thecells in Panel D were treated with M75 and immunogold only once they hadbeen embedded and sectioned, and thus antigens inside the cells couldalso be decorated.

FIG. 14 compares the results of immunizing baby rats to XC tumor cellswith rat serum prepared against the fusion protein MN glutathioneS-transferase (GEX-3X-MN) (the IM group) with the results of immunizingbaby rats with control rat sera (the C group). Each point on the graphrepresents the tumor weight of a tumor from one rat. Example 14 detailsthose experiments.

FIGS. 15A-C shows a complete nucleotide sequence of a MN cDNA [SEQ. ID.NO.: 5]. Also shown is the deduced amino acid sequence [SEQ. ID. NO.:6]. The polyadenylation signal (AATAAA) and the mRNA instability motif(ATTTA) are located at nucleotides (nts) 1507-1512 and at nts 1513-1518,respectively. The amino acid residues of the putative signal peptide aswell as the membrane-spanning segment are located at amino acids (aa)1-37 and at aa 415-433, respectively. The N-glycosylation site islocated at aa 346. The S/TPXX elements are located at amino acids 7-10,47-50, 71-74, 153-156, 162-165, 333-336, and 397-400.

FIG. 16 is a restriction map of the full-length MN cDNA. The openreading frame is shown as an open box. The thick lines below therestriction map illustrate the sizes and positions of two overlappingcDNA clones. The horizontal arrows indicate the positions of primers R1[SEQ. ID. NO.: 7] and R2 [SEQ. ID. NO.: 8] used for the 5′ end RACE.Relevant restriction sites are BamHI (B), EcoRV (V), EcoRI (E), PstI(Ps), PvuII (Pv).

FIG. 17 shows a restriction analysis of the MN gene. Genomic DNA fromHeLa cells was cleaved with the following restriction enzymes: EcoRI(1), EcoRV (2), HindIII (3), KpnI (4), NcoI (5), PstI (6), and PvuII(7), and then analyzed by Southern hybridization under stringentconditions using MN cDNA as a probe.

FIG. 18 provides a hydrophilicity profile of the MN protein shown inFIG. 15. The profile was computed using an average group length of 6amino acids.

FIG. 19( a) shows an alignment of HCA plots derived from MN, human CA VI(hCA) and CA II (CA2). A one-letter code is used for all amino acidswith exception of P (stars), G (diamond-shaped symbol), T and S (openand dotted squares, respectively). Strands D, E, F and G are essentialfor the structural core of CA. Topologically conserved hydrophobic aminoacids are shaded (in hCA VI and MN). Ligands of the catalytic zinc ion(His residues) are indicated by arrowheads.

FIG. 19( b) presents a stereoview of the CA II three-dimensionalstructure illustrating a superposition of the complete CA II structure(thin ribbon) with the structure which is well conserved in MN (openthick ribbon).

FIG. 19( c) presents an HCA comparison of thebasic/helix-loop-helix/zipper domains of Max and Myf-3 with theN-terminal part of MN. The 3D structure of Max is indicated above itsplot with delineation of the segments b: basic, h1: helix 1, L: loop,h2: helix 2, z: zipper. The I(6X)Y motif is shaded within helix 2.

FIG. 19( d) schematically represents the sequence elements andstructural domains predicted from the deduced amino acid sequence of MN.

FIG. 20 schematically represents an MN promoter region. The consensussequences are as follows: CAT—CCAAT; TATA—ATAAATATA; AP2-1—GSSWSCC;AP2—YSSCCMNSSS [SEQ. ID. NO.: 19]; SP1—KMGGCCKRRY [SEQ. ID. NO.: 20];p53—RRRCWWGYYY [SEQ. ID. NO.: 21]; and SDRE—CACCSCAC.

FIG. 21 schematically represents the 5′ MN genomic region of an MNgenomic clone.

FIG. 22( a) shows the zinc-binding activity of MN protein extracted fromHeLa cells persistently infected with LCMV. Samples were concentrated byimmunoprecipitation with Mab M75 before loading (A), and after elutionfrom ZnCl₂-saturated (B) or ZnCl₂-free Fast-Flow chelating Sepharasecolumn (c). Immunoprecipitates were analyzed by Western blotting usingiodinated M75 antibody.

FIG. 22 (b) shows MN protein binding to DNA-cellulose. Proteinsextracted from LCMV-infected HeLa cells were incubated withDNA-cellulose (A). Proteins that bound to DNA-cellulose in the presenceof ZnCl₂ and absence of DTT (B), in the presence of both ZnCl₂ and DTT(C), and in the absence of both ZnCl₂ and DTT (D) were eluted, and allsamples were analyzed as above.

FIG. 22( c) shows the results of endoglycosidase H and F digestion. MNprotein immunoprecipitated with Mab M75 was treated with Endo F (F) andEndo H(H). Treated (+) and control samples (−) were analyzed by Westernblotting as above.

FIG. 23 shows the morphology and growth kinetics of control (a, c, e andg) and MN-expressing (b, d, f and h) NIH 3T3 cells. The micrographs areof methanol fixed and Giemsa stained cells at a magnification X 100.Cells were grown to confluency (a, b), or as individual colonies inPetri dishes (c, d) and in soft agar (e, f). The (g) and (h) graphsprovide growth curves of cells cultured in DMEM medium containingrespectively, 10% and 1% FCS. The mean values of triplicatedeterminations were plotted against time.

FIG. 24 illustrates flow cytometric analyses of asynchronous cellpopulations of control and MN cDNA-transfected NIH 3T3 cells.

FIG. 25 a-b is the complete sequence of an MN genomic clone of thisinvention [SEQ. ID. NO.: 23]. It is 5052 nucleotides long with thetranscription start site at position 3534 (starting with ACAGTCA . . .). The presumed promoter region is about 300 to 400 nucleotides upstreamof the transcription start site.

DETAILED DESCRIPTION

As demonstrated herein MaTu was found to be a two-component system. Onepart of the complex, exogenous MX, is transmissible, and is manifestedby a protein, p58X, which is a cytoplasmic antigen which reacts withsome natural sera, of humans and of various animals. The othercomponent, MN, is endogenous to human cells. The level of MN expressionhas been found to be considerably increased in the presence of theMaTu-MX transmissible agent, which has been now identified aslymphocytic choriomeningitis virus (LCMV) which persistently infectsHeLa cells.

MN is a cellular gene, showing only very little homology with known DNAsequences. It is rather conservative and is present as a single copygene in the chromosomal DNA of various vertebrates. Described herein isthe cloning and sequencing of the MN cDNA, and the genetic engineeringof MN proteins—such as the GEX-3X-MN and MN 20-19 proteins. Therecombinant MN proteins can be conveniently purified by affinitychromatography.

MN is manifested in HeLa cells by a twin protein, p54/58N, that islocalized on the cell surface and in the nucleus. Immunoblots using amonoclonal antibody reactive with p54/58N (MAb M75) revealed two bandsat 54 kd and 58 kd. Those two bands may correspond to one type ofprotein that differs by glycosylation pattern or by how it is processed.(Both p54N and p58N are glycosylated with oligosaccharidic residuescontaining mannose, but only p58N also contains glucosamine.) Herein,the phrase “twin protein” indicates p54/58N.

MN is absent in rapidly growing, sparse cultures of HeLa, but isinducible either by keeping the cells in dense cultures or, moreefficiently, by infecting them with MX (LCMV). Those inducing factorsare synergistic. p54/58N and not p58X is associated with virions ofvesicular stomatitis virus (VSV), reproduced in MaTu-infected HeLa.Whereas the twin protein p54/58N is glycosylated and forms oligomerslinked by disulfidic bonds, p58X is not glycosylated and does not formS—S-linked oligomers.

VSV assembles p54/58N into virions in HeLa cells, indicating that thetwin protein is responsible for complementation of VSV G-protein mutantsand for formation of VSV(MaTu) pseudotypes. As only enveloped virusesprovide surface glycoproteins for the formation of infectious,functioning pseudotypes, which can perform such specific functions asadsorption and penetration of virions into cells [Zavada, J., J. Gen.Virol., 63: 15-24 (1982)], that observation implies that the MN genebehaves as a quasi-viral sequence.

The surface proteins of enveloped viruses, which participate in theformation of VSV pseudotypes, are glycosylated as is the MN twinprotein, p54/58N. MN proteins also resemble viral glycoproteins in theformation of oligomers (preferably tri- or tetramers); sucholigomerization, although not necessarily involving S—S bonds(disulfidic bonds), is essential for the assembly of virions [Kreis andLodish, Cell, 46: 929-937 (1986)]. The disulfidic bonds can be disruptedby reduction with 2-mercaptoethanol.

As reported in Pastorekova et al., Virology, 187: 620-626 (1992), afterreduction with mercaptoethanol, p54/58N from cell extracts or from VSVlooks very similar on immunoblot. Without reduction, in cell extracts,it gives several bands around 150 kd, suggesting that the cells maycontain several different oligomers (probably with a different p54:p58ratio), but VSV selectively assembles only one of them, with a molecularweight of about 153 kd. That oligomer might be a trimer, or rather atetramer, consisting of 54 kd and 58 kd proteins. The equimolar ratio ofp54:p58 in VSV virions is indicated by approximately the same strengthof 54 kd and 58 kd bands in a VSV sample analyzed under reducingconditions.

The expression of MN proteins appears to be diagnostic/prognostic forneoplastic disease. The MN twin protein, p54/58N, was found to beexpressed in HeLa cells and in Stanbridge's tumorigenic (H/F-T) hybridcells [Stanbridge et al., Somatic Cell Genet, 7: 699-712 (1981); andStanbridge et al., Science, 215: 252-259 (1982)] but not in fibroblastsor in non-tumorigenic (H/F-N) hybrid cells [Stanbridge et al., id.]. Inearly studies, MN proteins were found in immunoblots prepared from humanovarian, endometrial and uterine cervical carcinomas, and in some benignneoplasias (as mammary papilloma) but not from normal ovarian,endometrial, uterine or placental tissues. Example 13 details furtherresearch on MN gene expression wherein MN antigen, as detected byimmunohistochemical staining, was found to be prevalent in tumor cellsof a number of cancers, including cervical, bladder, head and neck, andrenal cell carcinomas among others. Further, the immunohistochemicalstaining experiments of Example 13 show that among normal tissuestested, only normal stomach tissues showed routinely and extensively thepresence of MN antigen. MN antigen is further shown herein to be presentsometimes in morphologically normal-appearing areas of tissue specimensexhibiting dysplasia and/or malignancy.

In HeLa cells infected with MX, observed were conspicuousultrastructural alterations, that is, the formation of abundantfilaments on cell surfaces and the amplification of mitochondria. Usingan immunogold technique, p54/58N was visualized on the surface filamentsand in the nucleus, particularly in the nucleoli. Thus MN proteinsappear to be strongly correlated with tumorigenicity, and do not appearto be produced in general by normal non-tumor cells.

Examples herein show that MX and MN are two different entities, that canexist independently of each other. MX (LCMV) as an exogenous,transmissible agent can multiply in fibroblasts and in H/F-N hybridcells which are not expressing MN-related proteins (FIG. 6). In suchcells, MX does not induce the production of MN protein. MN protein canbe produced in HeLa and other tumor cells even in the absence of MX asshown in FIGS. 6-9. However, MX is a potent inducer of MN-relatedprotein in HeLa cells; it increases its production thirty times over theconcentration observed in uninfected cells (FIGS. 7 and 12, Table 1 inExample 8, below).

MN Gene Cloning and Sequencing

FIGS. 1A-1B and 15 provide the nucleotide sequences for MN cDNA clonesisolated as described below, respectively SEQ. ID. NOS.: 1 and 5. FIG.25 a-b provides the sequence of a MN genomic clone containing a promoterregion [SEQ. ID. NO.: 23].

It is understood that because of the degeneracy of the genetic code,that is, that more than one codon will code for one amino acid [forexample, the codons TTA, TTG, CTT, CTC, CTA and CTG each code for theamino acid leucine (leu)], that variations of the nucleotide sequencesin, for example, SEQ. ID. NOS.: 1, 5, and 23 wherein one codon issubstituted for another, would produce a substantially equivalentprotein or polypeptide according to this invention. All such variationsin the nucleotide sequences of the MN cDNA and complementary nucleicacid sequences are included within the scope of this invention.

It is further understood that the nucleotide sequences herein describedand shown in FIGS. 1A-1B, 15 and 25, represent only the precisestructures of the cDNA and genomic nucleotide sequences isolated anddescribed herein. It is expected that slightly modified nucleotidesequences will be found or can be modified by techniques known in theart to code for substantially similar or homologous MN proteins andpolypeptides, for example, those having similar epitopes, and suchnucleotide sequences and proteins/polypeptides are considered to beequivalents for the purpose of this invention. DNA or RNA havingequivalent codons is considered within the scope of the invention, asare synthetic nucleic acid sequences that encode proteins/polypeptideshomologous or substantially homologous to MN proteins/polypeptides, aswell as those nucleic acid sequences that would hybridize to saidexemplary sequences [SEQ. ID. NOS. 1, 5 and 23] under stringentconditions or that but for the degeneracy of the genetic code wouldhybridize to said cDNA nucleotide sequences under stringenthybridization conditions. Modifications and variations of nucleic acidsequences as indicated herein are considered to result in sequences thatare substantially the same as the exemplary MN sequences and fragmentsthereof.

Partial cDNA Clone

To find the MN gene, a lambda gt11 cDNA library from MX-infected HeLacells was prepared. Total RNA from MX-infected HeLa cells was isolatedby a guanidinium-thiocyanate-CsCl method [Chirgwin et al., Biochemistry,18: 5249 (1979)], and the mRNA was affinity separated on oligodT-cellulose [Ausubel et al., Short Protocols in Molecular Biology,(Greene Publishing Assocs. and Wiley-Interscience; NY, USA, 1989]. Thesynthesis of the cDNA and its cloning into lambda gt11 was carried outusing kits from Amersham, except that the EcoRI-NotI adaptor was fromStratagene [La Jolla, Calif. (USA)]. The library was subjected toimmunoscreening with Mab M75 in combination with goat anti-mouseantibodies conjugated with alkaline phosphatase. That immunoscreeningmethod is described in Young and Davis, PNAS (USA), 80: 1194-1198(1983). About 4×10⁵ primary plaques on E. coli Y1090 cells, representingabout one-half of the whole library, were screened using Hybond N+membrane [Amersham] saturated with 10 mM IPTG and blocked with 5% FCS.Fusion proteins were detected with Mab M75 in combination with goatanti-mouse antibodies conjugated with alkaline phosphatase. One positiveclone was picked.

pBluescript-MN. The positive clone was subcloned into the NotI site ofpBluescript KS [Stratagene] thereby creating pBluescript-MN. Twooppositely oriented nested deletions were made using Erase-a-Base™ kit[Promega; Madison, Wis. (USA)] and sequenced by dideoxy method with a T7sequencing kit [Pharmacia; Piscataway, N.J. (USA)]. The sequencingshowed a partial cDNA clone, the insert being 1397 bp long. Thatsequence is shown in FIG. 1A-1B. The sequence comprise a large 1290 bpopen reading frame and 107 bp 3′ untranslated region containing apolyadenylation signal (AATAAA). Another interesting feature of thesequence is the presence of a region contributing to instability of themRNA (AUUUA at position 1389) which is characteristic for mRNAs of someoncogenes and lymphokines [Shaw and Kamen, Cell, 46: 659-667 (1986)].However, the sequence surrounding the first ATG codon in the openreading frame (ORF) did not fit the definition of a translational startsite. In addition, as follows from a comparison of the size of the MNclone with that of the corresponding mRNA in a Northern blot (FIG. 4),the cDNA was missing about 100 bp from the 5′ end of its sequence.

Full-Length cDNA Clone

Attempts to isolate a full-length clone from the original cDNA libraryfailed. Therefore, we performed a rapid amplification of cDNA ends(RACE) using MN-specific primers, R1 and R2, derived from the 5′ regionof the original cDNA clone. The RACE product was inserted intopBluescript, and the entire population of recombinant plasmids wassequenced with an MN-specific primer ODN1. In that way, we obtained areliable sequence at the very 5′ end of the MN cDNA as shown in FIG. 15[SEQ. ID. NO.: 5].

Specifically, RACE was performed using 5′ RACE System [GIBCO BRL;Gaithersburg, Md. (USA)] as follows. 1 μg of mRNA (the same as above)was used as a template for the first strand cDNA synthesis which wasprimed by the MN-specific antisense oligonucleotide, R1(5′-TGGGGTTCTTGAGGATCTCCAGGAG-3′) [SEQ. ID. NO.: 7]. The first strandproduct was precipitated twice in the presence of ammonium acetate and ahomopolymeric C tail was attached to its 3′ end by TdT. Tailed cDNA wasthen amplified by PCR using a nested primer, R2(5′-CTCTAACTTCAGGGAGCCCTCTTCTT-3′) [SEQ. ID. NO.: 8] and an anchorprimer that anneals to the homopolymeric tail(5′-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGI IGGGIIGGGIIG-3′) [SEQ. ID. NO.:9]. Amplified product was digested with BamHI and SalI restrictionenzymes and cloned into pBluescript II KS plasmid. After transformation,plasmid DNA was purified from the whole population of transformed cellsand used as a template for the sequencing with the MN-specific primerODN1 [SEQ. ID. NO.: 3; a 29-mer, the sequence for which is shown inExample 10].

The full-length MN cDNA sequence is 1519 base pairs (bp) long (FIG. 15).It contains a single ORF of 1400 bp, starting at position 12, with anATG codon that is in a good context (GCGCATGG) with the rule proposedfor translation initiation [Kozak, J. Cell. Biol., 108: 229-241 (1989)].The AT rich 3′ untranslated region contains, as indicated above, apolyadenylation signal (AATAAA) preceding the end of the cDNA by 10 bp.Surprisingly, the sequence from the original clone as well as from fouradditional clones obtained from the same cDNA library did not reveal anypoly(A) tail. Moreover, also as indicated above, just downstream of thepoly(A) signal we found an ATTTA motif that is thought to contribute tomRNA instability (Shaw and Kamen, supra). This fact raised possibilitythat the poly (A) tail is missing due to the specific degradation of theMN mRNA.

Genomic Clone

To study MN regulation, an MN genomic clone was isolated from a humancosmid library prepared from fetal brain using both the MN cDNA probeand the MN-specific primers derived from the 5′ end of the cDNA [SEQ.ID. NOS.: 3 and 4; ODN1 AND ODN2; see Example 10]. The sequence for thatgenomic clone is shown in FIG. 25 [SEQ. ID. NO.: 23]. Sequence analysisrevealed that the genomic clone covers a region upstream from the MNtranscription start site and ending with the BamHI restriction sitelocalized inside the MN cDNA. Other MN genomic clones can be similarlyisolated.

The promoter region is GC-rich and contains one putative TATA-box 578 bpupstream from the transcription start. The promoter contains severalconsensus sequences for binding sites of regulatory elements, includingtwo p53 sites, two AP-2 sites, an AP-1 site, a SP-1 site, and a SDREsite. FIG. 20 provides a schematic of the MN promoter region, and FIG.21 provides a schematic of the 5′ MN genomic region.

Interestingly, the 5′ end region of the isolated genomic clone isstrongly homologous to the 5′ long terminal repeat (LTR) of humanendogenous retroviruses HERV K. As shown in FIG. 21, there is no codingsequence, only two Alu repeats, between the LTR-like region and thepromoter. That fact suggests that the LTR, although not necessarilybelonging to MN, may provide an enhancer for MN transcription.

Deduced Amino Acid Sequences

The open reading frame of the MN cDNA clone shown in FIG. 1A-1B encodesa putative protein of 429 amino acids with a calculated molecular weightof about 48 kd. The hydrophilicity profile reveals a hydrophobicsequence of amino acids (at positions 371-395) probably representing theregion spanning the plasma membrane and containing also a potentialcleavage signal. The profile fits well with the observation that p54/58Nproteins are localized on the cell membrane. There are no PEST regionsin the MN amino acid sequence, suggesting that the product of the MNgene is a stable long-lived protein [Rogers et al., Science, 234:364-368 (1986)]. Such a feature explains our experience with inefficientmetabolic labeling of p54/58N. The deduced amino acid sequence displaysalso other features namely, 10 potential phosphorylation and 7myristylation sites, and 3 antigenic determinants.

The deduced amino acid sequence from the partial cDNA sequence shown inFIG. 1A-1B can be compared to that shown in FIG. 15 from the full-lengthcDNA. The partial sequence is missing the N-terminal 37 amino acids—theputative signal peptide. The ORF of the MN cDNA shown in FIG. 15 has thecoding capacity for a 466 amino acid protein with a calculated molecularweight of 51.5 kd. As assessed by amino acid sequence analysis, thededuced primary structure of the MN protein can be divided into fourdistinct regions. The initial hydrophobic region of 37 amino acids (AA)corresponds to a signal peptide. The mature protein has an N-terminalpart of 377 AA, a hydrophobic transmembrane segment of 20 AA and aC-terminal region of 32 AA. The overall amino acid composition is ratherbasic, with a predicted isoelectric point of 8.92. The MN protein isrich in leucine (11.16%), proline (10.3%), alanine (9.44%), arginine(9.23%), and serine (9.01%).

More detailed insight into MN protein primary structure disclosed thepresence of several consensus sequences. One potential N-glycosylationsite was found at position 345 of FIG. 15, and a putative nuclearlocalization signal composed of a stretch of basic amino acids RRARKK[Blank et al., EMBO J., 10: 4159-4167 (1991); Wang and Reed, Nature,364: 121-126 (1993)] was recognized in the middle of the protein, atposition 279-284 of FIG. 15. These features, together with the predictedmembrane-spanning region mentioned above, are consistent with theresults, in which MN was shown to be an N-glycosylated protein localizedboth in the plasma membrane and in the nucleus. MN protein sequencededuced from cDNA was also found to contain six S/TPXX sequence elements[SEQ. ID. NOS.: 25 AND 26] (one of them is in the signal peptide)defined by Suzuki, J. Mol. Biol., 207: 61-84 (1989) as motifs frequentlyfound in gene regulatory proteins. However, only two of them arecomposed of the suggested consensus amino acids.

Sequence Similarities and HCA

Computer analysis of the MN cDNA sequence was carried out using DNASISand PROSID (Pharmacia Software packages). GenBank, EMBL, ProteinIdentification Resource and SWISS-PROT databases were searched for allpossible sequence similarities. In addition, a search for proteinssharing sequence similarities with MN was performed in the MIPS databankwith the FastA program [Pearson and Lipman, PNAS (USA), 85: 2444(1988)].

The MN gene was found to clearly be a novel sequence derived from thehuman genome. Searches for amino acid sequence similarities in proteindatabases revealed as the closest homology a level of sequence identity(44% of 170 AAs) between the central part of the MN protein [AAs 221-390of FIG. 15 (SEQ. ID. NO.: 6)] and carbonic anhydrases (CA). However, theoverall sequence homology between the cDNA MN sequence and cDNAsequences encoding different CA isoenzymes is in a homology range of48-50% which is considered by ones in the art to be low. Therefore, theMN cDNA sequence is not closely related to any CA cDNA sequences.

Only very closely related sequences having a homology of at least 80-90%would hybridize to each other under stringent conditions. A sequencecomparison of the MN cDNA sequence shown in FIG. 1A-1B and acorresponding cDNA of the human carbonic anhydrase II (CA II) showedthat there are no stretches of identity between the two sequences thatwould be long enough to allow for a segment of the CA II cDNA sequencehaving 50 or more nucleotides to hybridize under stringent hybridizationconditions to the MN cDNA or vice versa.

Although MN deduced amino acid sequences show some homology to knowncarbonic anhydrases, they differ from them in several respects. Sevencarbonic anhydrases are known [Dodgson et al. (eds.), The CarbonicAnhydrases, (Plenum Press; New York/London (1991)]. All of the knowncarbonic anhydrases are proteins of about 30 kd, smaller than thep54/58N-related products of the MN gene. Further, the carbonicanhydrases do not form oligomers as do the MN-related proteins.

HCA. Hydrophobic Cluster Analysis (HCA) was used as a very sensitivemethod to detect similarities in the secondary and tertiary folding ofprotein domains even if the sequence homology is low. [Lemesle-Varlootet al., Biochimie, 72: 555 (1990); Thoreau et al., FEBS Lett., 282: 26(1991); Gaboriaud et al., FEBS Lett., 224: 189 (1987).] Comparison ofthe HCA plots for MN, human CA VI and CA II [FIG. 19( a)] showed thatonly the middle and C-terminal part of CA are highly conserved in MNwith the conserved zinc binding site and the enzyme's active center. A44% sequence identity and 87% HCA score was calculated between MN and CAVI isoenzyme. [Aldred et al., Biochemistry, 30: 569 (1991).] Thosevalues are within the range of those observed for closely relatedstructures [Lemesle-Varloot et al., supra].

HCA was also used in combination with the one-dimensional analysisprograms, to analyze the N-terminal part of MN. When screened againstthe MIPS sequence databank using the FastA program [Pearson and Lipman,supra], the N-terminal segment of MN (AAs 38-114 of SEQ. ID. NO.: 6)repeatedly matched helical protein domains involved in the regulation ofgene expression (i.e., transforming protein Myb, accession numberS04897; myogenic determination factor Myf-3, accession number S06947;transcriptional activator YAB, accession number JE0416; translationalactivator PET127, accession number S17029, etc.). HCA suggested [FIG.19( c)] that although those hits were of low level sequence identity,they might be structurally valid.

The most significant is a predicted structural similarity of MN tomembers of the HLH protein family—represented by Myf-3 and Max protein.The conserved I(6X)Y motif is revealed in FIG. 19( c), a motif that is acommon characteristic of an HLH protein dimerization domain[Ferre-D'Amare et al., Nature, 363: 38 (1993)]. The region between theCA-like domain and the putative HLH (covering AAs 120-220 of SEQ. ID.NO.: 6) domain is rich in imperfect repeats of Ser (15%), Pro (16%), Glyand acidic residues with few hydrophobic amino acids, resembling, thus,an activation region of transcription factors. [Lautenberger et al.,Oncogene, 7: 1713 (1992).]

In experiments, the results for which are shown in FIG. 22( a), it wasdetermined that MN protein is able to bind zinc cations, as shown byaffinity chromatography using Zn-charged chelating sepharose. MN proteinimmunoprecipitated from HeLa cells by Mab M75 was found to have weakcatalytic activity of CA. The CA-like domain of MN has a structuralpredisposition to serve as a binding site for small soluble domains.Thus, MN protein could mediate some kind of signal transduction.

MN protein from LCMV-infected HeLA cells was shown by using DNAcellulose affinity chromatography [FIG. 22( b)] to bind to immobilizeddouble-stranded salmon sperm DNA. The binding activity required both thepresence of zinc cations and the absence of a reducing agent in thebinding buffer.

MN Twin Protein

The possibility that the 4 kd difference between the molecular weightsof the two MN proteins is caused by different glycosylation was ruledout, since after in vitro treatment with endoglycosidases H and F,respectively, both peptide portions lost about 3 kd in weight. Thisresult indicates, in addition, that the molecular weight of the smaller54 kd MN protein without its 3 kd sugar moiety, roughly corresponds tothe molecular weight of MN calculated from the full-length cDNA. Westernblot analysis of MN proteins from cervical carcinoma and normal stomachshows that in both tissues MN protein consists of two 54 and 58 kdpeptide portions.

To determine whether both p54/58N proteins were encoded by one gene,antisense ODNs were used to inhibit specifically MN gene expression.[Such use of antisense ODNs is reviewed in Stein and Cohen, Cancer Res.,48: 2659-2668 (1988).] Those experiments are detailed in Example 10. Thefindings indicated that cultivation of HeLa cells with ODNs resulted ina considerable inhibition of p54/58N synthesis, whereas the amount ofdifferent HeLa cell proteins produced remained approximately the same.Further, and importantly, on immunoblotting, the specific inhibition byODNs affected both of the p54/58N proteins (FIG. 3). Thus, it wasconcluded that the MN gene that was cloned codes for both of the p54/58Nproteins in HeLa cells.

MN Proteins and/or Polypeptides

The phrase “MN proteins and/or polypeptides” (MN proteins/polypeptides)is herein defined to mean proteins and/or polypeptides encoded by an MNgene or fragments thereof. Exemplary and preferred MN proteins accordingto this invention have the deduced amino acid sequences shown in FIGS.1A-1B and 15. Preferred MN proteins/polypeptides are those proteinsand/or polypeptides that have substantial homology with the MN proteinsshown in FIGS. 1A-1B and 15. For example, such substantially homologousMN proteins/polypeptides are those that are reactive with theMN-specific antibodies of this invention, preferably the Mabs M75, MN12and MN7 or their equivalents.

A “polypeptide” is a chain of amino acids covalently bound by peptidelinkages and is herein considered to be composed of 50 or less aminoacids. A “protein” is herein defined to be a polypeptide composed ofmore than 50 amino acids.

MN proteins exhibit several interesting features: cell membrane, and atthe same time, nuclear localization (similar to E6 protein of HPV16),cell density dependent expression in HeLa cells, correlation with thetumorigenic phenotype of HeLa×fibroblast somatic cell hybrids, andexpression in several human carcinomas among other tissues. Asdemonstrated herein, for example, in Example 13, MN protein can be founddirectly in tumor tissue sections but not in general in counterpartnormal tissues (exceptions noted infra in Example 13 as in normalstomach tissues). MN is also expressed sometimes in morphologicallynormal appearing areas of tissue specimens exhibiting dysplasia and/ormalignancy. Taken together, these features suggest a possibleinvolvement of MN in the regulation of cell proliferation,differentiation and/or transformation.

It can be appreciated that a protein or polypeptide produced by aneoplastic cell in vivo could be altered in sequence from that producedby a tumor cell in cell culture or by a transformed cell. Thus, MNproteins and/or polypeptides which have varying amino acid sequencesincluding without limitation, amino acid substitutions, extensions,deletions, truncations and combinations thereof, fall within the scopeof this invention. It can also be appreciated that a protein extantwithin body fluids is subject to degradative processes, such as,proteolytic processes; thus, MN proteins that are significantlytruncated and MN polypeptides may be found in body fluids, such as,sera. The phrase “MN antigen” is used herein to encompass MN proteinsand/or polypeptides.

It will further be appreciated that the amino acid sequence of MNproteins and polypeptides can be modified by genetic techniques. One ormore amino acids can be deleted or substituted. Such amino acid changesmay not cause any measurable change in the biological activity of theprotein or polypeptide and result in proteins or polypeptides which arewithin the scope of this invention.

The MN proteins and polypeptides of this invention can be prepared in avariety of ways according to this invention, for example, recombinantly,synthetically or otherwise biologically, that is, by cleaving longerproteins and polypeptides enzymatically and/or chemically. A preferredmethod to prepare MN proteins is by a recombinant means. Particularlypreferred methods of recombinantly producing MN proteins are describedbelow for the GEX-3X-MN and MN 20-19 proteins.

Recombinant Production of MN Proteins and Polypeptides

A representative method to prepare the MN proteins shown in FIGS. 1A-1Band 15 or fragments thereof would be to insert the appropriate fragmentof MN cDNA into an appropriate expression vector as exemplified below.The fusion protein GEX-3X-MN expressed from XL1-Blue cells isnonglycosylated. Representative of a glycosylated, recombinantlyproduced MN protein is the MN 20-19 protein expressed from insect cells.The MN 20-19 protein was also expressed in a nonglycosylated form in E.coli using the expression plasmid pEt-22b [Novagen].

Baculovirus Expression Systems. Recombinant baculovirus express vectorshave been developed for infection into several types of insect cells.For example, recombinant baculoviruses have been developed for amongothers: Aedes aegypti, Autographa californica, Bombyx mor, Drosphilamelanogaster, Heliothis zea, Spodoptera frugiperda, and Trichoplusia ni[PCT Pub. No. WO 89/046699; Wright, Nature, 321: 718 (1986); Fraser etal., In Vitro Cell Dev. Biol., 25: 225 (1989). Methods of introducingexogenous DNA into insect hosts are well-known in the art. DNAtransfection and viral infection procedures usually vary with the insectgenus to be transformed. See, for example, Autographa [Carstens et al.,Virology, 101: 311 (1980)]; Spodoptera [Kang, “Baculovirus Vectors forExpression of Foreign Genes,” in: Advances in Virus Research, 35(1988)]; and Heliothis (virescens) [PCT Pub. No. WO 88/02030].

A wide variety of other host-cloning vector combinations may be usefullyemployed in cloning the MN DNA isolated as described herein. Forexample, useful cloning vehicles may include chromosomal, nonchromosomaland synthetic DNA sequences such as various known bacterial plasmidssuch as pBR322, other E. coli plasmids and their derivatives and widerhost range plasmids such as RP4, phage DNA, such as, the numerousderivatives of phage lambda, e.g., NB989 and vectors derived fromcombinations of plasmids and phage DNAs such as plasmids which have beenmodified to employ phage DNA expression control sequences.

Useful hosts may be eukaryotic or prokaryotic and include bacterialhosts such as E. coli and other bacterial strains, yeasts and otherfungi, animal or plant hosts such as animal or plant cells in culture,insect cells and other hosts. Of course, not all hosts may be equallyefficient. The particular selection of host-cloning vehicle combinationmay be made by those of skill in the art after due consideration of theprinciples set forth herein without departing from the scope of thisinvention.

The particular site chosen for insertion of the selected DNA fragmentinto the cloning vehicle to form a recombinant DNA molecule isdetermined by a variety of factors. These include size and structure ofthe protein or polypeptide to be expressed, susceptibility of thedesired protein or polypeptide to endoenzymatic degradation by the hostcell components and contamination by its proteins, expressioncharacteristics such as the location of start and stop codons, and otherfactors recognized by those of skill in the art.

The recombinant nucleic acid molecule containing the MN gene, fragmentthereof, or cDNA therefrom, may be employed to transform a host so as topermit that host (transformant) to express the structural gene orfragment thereof and to produce the protein or polypeptide for which thehybrid DNA encodes. The recombinant nucleic acid molecule may also beemployed to transform a host so as to permit that host on replication toproduce additional recombinant nucleic acid molecules as a source of MNnucleic acid and fragments thereof. The selection of an appropriate hostfor either of those uses is controlled by a number of factors recognizedin the art. These include, for example, compatibility with the chosenvector, toxicity of the co-products, ease of recovery of the desiredprotein or polypeptide, expression characteristics, biosafety and costs.

Where the host cell is a procaryote such as E. coli, competent cellswhich are capable of DNA uptake are prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ method bywell known procedures. Transformation can also be performed afterforming a protoplast of the host cell.

Where the host used is an eukaryote, transfection methods such as theuse of a calcium phosphate precipitate, electroporation, conventionalmechanical procedures such as microinjection, insertion of a plasmidencapsulated in red blood cell ghosts or in liposomes, treatment ofcells with agents such as lysophosphatidyl-choline or use of virusvectors, or the like may be used.

The level of production of a protein or polypeptide is governed by threemajor factors: (1) the number of copies of the gene or DNA sequenceencoding for it within the cell; (2) the efficiency with which thosegene and sequence copies are transcribed and translated; and (3) thestability of the mRNA. Efficiencies of transcription and translation(which together comprise expression) are in turn dependent uponnucleotide sequences, normally situated ahead of the desired codingsequence. Those nucleotide sequences or expression control sequencesdefine, inter alia, the location at which an RNA polymerase interacts toinitiate transcription (the promoter sequence) and at which ribosomesbind and interact with the mRNA (the product of transcription) toinitiate translation. Not all such expression control sequences functionwith equal efficiency. It is thus of advantage to separate the specificcoding sequences for the desired protein from their adjacent nucleotidesequences and fuse them instead to known expression control sequences soas to favor higher levels of expression. This having been achieved, thenewly engineered DNA fragment may be inserted into a multicopy plasmidor a bacteriophage derivative in order to increase the number of gene orsequence copies within the cell and thereby further improve the yield ofexpressed protein.

Several expression control sequences may be employed. These include theoperator, promoter and ribosome binding and interaction sequences(including sequences such as the Shine-Dalgarno sequences) of thelactose operon of E. coli (“the lac system”), the correspondingsequences of the tryptophan synthetase system of E. coli (“the trpsystem”), a fusion of the trp and lac promoter (“the tac system”), themajor operator and promoter regions of phage lambda (O_(L)P_(L) andO_(R)P_(R),), and the control region of the phage fd coat protein. DNAfragments containing these sequences are excised by cleavage withrestriction enzymes from the DNA isolated from transducing phages thatcarry the lac or trp operons, or from the DNA of phage lambda or fd.Those fragments are then manipulated in order to obtain a limitedpopulation of molecules such that the essential controlling sequencescan be joined very close to, or in juxtaposition with, the initiationcodon of the coding sequence.

The fusion product is then inserted into a cloning vehicle fortransformation or transfection of the appropriate hosts and the level ofantigen production is measured. Cells giving the most efficientexpression may be thus selected. Alternatively, cloning vehiclescarrying the lac, trp or lambda P_(L) control system attached to aninitiation codon may be employed and fused to a fragment containing asequence coding for a MN protein or polypeptide such that the gene orsequence is correctly translated from the initiation codon of thecloning vehicle.

The phrase “recombinant nucleic acid molecule” is herein defined to meana hybrid nucleotide sequence comprising at least two nucleotidesequences, the first sequence not normally being found together innature with the second.

The phrase “expression control sequence” is herein defined to mean asequence of nucleotides that controls and regulates expression ofstructural genes when operatively linked to those genes.

The following are representative examples of genetically engineering MNproteins of this invention. The descriptions are exemplary and not meantto limit the invention in any way.

Production of Fusion Protein GEX-3X-MN

To confirm whether the partial cDNA clone codes for the p54/58N-specificprotein, it was subcloned into the bacterial expression vector pGEX-3X[Pharmacia; Upsala, Sweden], constructed to express a fusion proteincontaining the C-terminus of glutathione S-transferase. The partial cDNAinsert from the above-described pBluescript-MN was released by digestingthe plasmid DNA by NotI. It was then treated with S1 nuclease to obtainblunt ends and then cloned into a dephosphorylated SmaI site of pGEX-3X(Pharmacia). After transformation of XL1-Blue cells [E. coli strain;Stratagene] and induction with IPTG, a fusion protein was obtained.

The fusion protein—MN glutathione S-transferase (GEX-3X-MN) was purifiedby affinity chromatography on Glutathione-S-Sepharose 4B [Pharmacia].Twenty micrograms of the purified recombinant protein in each of twoparallel samples were separated by SDS-PAGE on a 10% gel. One of thesamples (A) was stained with Coomassie brilliant blue, whereas the other(B) was blotted onto a Hybond C membrane [Amersham]. The blot wasdeveloped by autoradiography with ¹²⁵I-labeled MAb M75. The results areshown in FIG. 2.

SDS-PAGE analysis provided an interesting result: a number of proteinbands with different molecular weights (FIG. 2A). A similar SDS-PAGEpattern was obtained with another representative fusion protein producedaccording to this invention, beta-galactosidase-MN that was expressedfrom lambda gt11 lysogens. It appears that those patterns are due totranslation errors caused by the presence of 9 AGGAGG codon tandems inthe MN sequence. The use of those codons is strongly avoided inbacterial genes because of the shortage of corresponding tRNAs. Thus,during the translation of AGGAGG tandems from foreign mRNA, +1 ribosomalframeshifts arise with a high frequency (about 50%) [Spanjaard et al.,Nuc. Acid Res., 18: 5031-5036 (1990)].

By immunoblotting, a similar pattern was obtained: all the bands seen onstained SDS-PAGE gel reacted with the MN-specific MAb M75 (FIG. 2B),indicating that all the protein bands are MN-specific. Also, that resultindicates that the binding site for MAb M75 is on the N-terminal part ofthe MN protein, which is not affected by frameshifts.

As shown in Example 8 below, the fusion protein GEX-3X-MN was used inradioimmunoassays for MN-specific antibodies and for MN antigen.

Expression of MN 20-19 Protein

Another representative, recombinantly produced MN protein of thisinvention is the MN 20-19 protein which, when produced inbaculovirus-infected Sf9 cells [Spodoptera frugiperda cells; Clontech;Palo Alto, Calif. (USA)], is glycosylated. The MN 20-19 protein missesthe putative signal peptide (AAs 1-37) of SEQ. ID. NO.: 6 (FIG. 15), hasa methionine (Met) at the N-terminus for expression, and aLeu-Glu-His-His-His-His-His-His [SEQ. ID NO.: 22] added to theC-terminus for purification. In order to insert the portion of the MNcoding sequence for the GEX-3X-MN fusion protein into alternateexpression systems, a set of primers for PCR was designed. The primerswere constructed to provide restriction sites at each end of the codingsequence, as well as in-frame start and stop codons. The sequences ofthe primers, indicating restriction enzyme cleavage sites and expressionlandmarks, are shown below.

The SEQ. ID. NOS.: 17 and 18 primers were used to amplify the MN codingsequence present in the pGEX-3X-MN vector using standard PCR techniques.The resulting PCR product (termed MN 20-19) was electrophoresed on a0.5% agarose/1×TBE gel; the 1.3 kb band was excised; and the DNArecovered using the Gene Clean II kit according to the manufacturer'sinstructions [Bio101; LaJolla, Calif. (USA)].

MN 20-19 and plasmid pET-22b [Novagen, Inc.; Madison, Wis. (USA)] werecleaved with the restriction enzymes NdeI and XhoI, phenol-chloroformextracted, and the appropriate bands recovered by agarose gelelectrophoresis as above. The isolated fragments were ethanolco-precipitated at a vector:insert ratio of 1:4. After resuspension, thefragments were ligated using T4 DNA ligase. The resulting product wasused to transform competent Novablue E. coli cells [Novagen, Inc.].Plasmid mini-preps [Magic Minipreps; Promega] from the resultantampicillin resistant colonies were screened for the presence of thecorrect insert by restriction mapping. Insertion of the gene fragmentinto the pET-22b plasmid using the NdeI and XhoI sites added a6-histidine tail to the protein that could be used for affinityisolation.

To prepare MN 20-19 for insertion into the baculovirus expressionsystem, the MN 20-19 gene fragment was excised from pET-22b using therestriction endonucleases XbaI and PvuI. The baculovirus shuttle vectorpBacPAK8 [Clontech] was cleaved with XbaI and PacI. The desiredfragments (1.3 kb for MN 20-19 and 5.5 kb for pBacPAK8) were isolated byagarose gel electrophoresis, recovered using Gene Clean II, andco-precipitated at an insert:vector ratio of 2.4:1.

After ligation with T4 DNA ligase, the DNA was used to transformcompetent NM522 E. coli cells (Stratagene). Plasmid mini-preps fromresultant ampicillin resistant colonies were screened for the presenceof the correct insert by restriction mapping. Plasmid DNA from anappropriate colony and linearized BacPAK6 baculovirus DNA [Clontech]were used to transform Sf9 cells by standard techniques. Recombinationproduced BacPAK viruses carrying the MN 20-19 sequence. Those viruseswere plated onto Sf9 cells and overlaid with agar.

Plaques were picked and plated onto Sf9 cells. The conditioned media andcells were collected. A small aliquot of the conditioned media was setaside for testing. The cells were extracted with PBS with 1% TritonX100.

The conditioned media and the cell extracts were dot blotted ontonitrocellulose paper. The blot was blocked with 5% non-fat dried milk inPBS. Mab M75 were used to detect the MN 20-19 protein in the dot blots.A rabbit anti-mouse Ig-HRP was used to detect bound Mab M75. The blotswere developed with TMB/H₂0₂ with a membrane enhancer [KPL;Gaithersburg, Md. (USA)]. Two clones producing the strongest reaction onthe dot blots were selected for expansion. One was used to produce MN20-19 protein in High Five cells [Invitrogen Corp., San Diego, Calif.(USA); BTI-TN-5BI-4; derived from Trichoplusia ni egg cell homogenate].MN 20-19 protein was purified from the conditioned media from the virusinfected High Five cells.

The MN 20-19 protein was purified from the conditioned media byimmunoaffinity chromatography. 6.5 mg of Mab M75 was coupled to 1 g ofTresyl activated Toyopearl™ [solid support in bead form; Tosoh, Japan(#14471)]. Approximately 150 ml of the conditioned media was run throughthe M75-Toyopearl column. The column was washed with PBS, and the MN20-19 protein was eluted with 1.5 M MgCl. The eluted protein was thendialyzed against PBS.

Synthetic and Biologic Production of MN Proteins and Polypeptides

MN proteins and polypeptides of this invention may be prepared not onlyby recombinant means but also by synthetic and by other biologic means.Synthetic formation of the polypeptide or protein requires chemicallysynthesizing the desired chain of amino acids by methods well known inthe art. Exemplary of other biologic means to prepare the desiredpolypeptide or protein is to subject to selective proteolysis a longerMN polypeptide or protein containing the desired amino acid sequence;for example, the longer polypeptide or protein can be split withchemical reagents or with enzymes.

Chemical synthesis of a peptide is conventional in the art and can beaccomplished, for example, by the Merrifield solid phase synthesistechnique [Merrifield, J., Am. Chem. Soc., 85: 2149-2154 (1963); Kent etal., Synthetic Peptides in Biology and Medicine, 29 f.f. eds. Alitalo etal., (Elsevier Science Publishers 1985); and Haug, J. D., “PeptideSynthesis and Protecting Group Strategy”, American BiotechnologyLaboratory, 5(1): 40-47 (January/February 1987)].

Techniques of chemical peptide synthesis include using automatic peptidesynthesizers employing commercially available protected amino acids, forexample, Biosearch [San Rafael, Calif. (USA)] Models 9500 and 9600;Applied Biosystems, Inc. [Foster City, Calif. (USA)] Model 430; Milligen[a division of Millipore Corp.; Bedford, Mass. (USA)] Model 9050; and DuPont's RAMP (Rapid Automated Multiple Peptide Synthesis) [Du PontCompass, Wilmington, Del. (USA)].

Regulation of MN Expression and MN Promoter

MN appears to be a novel regulatory protein that is directly involved inthe control of cell proliferation and in cellular transformation. InHeLa cells, the expression of MN is positively regulated by celldensity. Its level is increased by persistent infection with LCMV. Inhybrid cells between HeLa and normal fibroblasts, MN expressioncorrelates with tumorigenicity. The fact that MN is not present innontumorigenic hybrid cells (CGL1), but is expressed in a tumorigenicsegregant lacking chromosome 11, indicates that MN is negativelyregulated by a putative suppressor in chromosome 11.

Evidence supporting the regulatory role of MN protein was found in thegeneration of stable transfectants of NIH 3T3 cells that constitutivelyexpress MN protein as described in Example 15. As a consequence of MNexpression, the NIH 3T3 cells acquired features associated with atransformed phenotype: altered morphology, increased saturation density,proliferative advantage in serum-reduced media, enhanced DNA synthesisand capacity for anchorage-independent growth. Further, as shown inExample 16, flow cytometric analyses of asynchronous cell populationsindicated that the expression of MN protein leads to acceleratedprogression of cells through G1 phase, reduction of cell size and theloss of capacity for growth arrest under inappropriate conditions. Also,Example 16 shows that MN expressing cells display a decreasedsensitivity to the DNA damaging drug mitomycin C.

Nontumorigenic human cells, CGL1 cells, were also transfected with thefull-length MN cDNA. The same pSG5C-MN construct in combination withpSV2neo plasmid as used to transfect the NIH 3T3 cells (Example 15) wasused. Also the protocol was the same except that the G418 concentrationwas increased to 1000 μg/ml.

Out of 15 MN-positive clones (tested by SP-RIA and Western blotting), 3were chosen for further analysis. Two MN-negative clones isolated fromCGL1 cells transfected with empty plasmid were added as controls.Initial analysis indicates that the morphology and growth habits ofMN-transfected CGL1 cells are not changed dramatically, but theirproliferation rate and plating efficiency is increased.

MN cDNA and promoter. When the promoter region from the MN genomicclone, isolated as described above, was linked to MN cDNA andtransfected into CGL1 hybrid cells, expression of MN protein wasdetectable immediately after selection. However, then it graduallyceased, indicating thus an action of a feedback regulator. The putativeregulatory element appeared to be acting via the MN promoter, becausewhen the full-length cDNA (not containing the promoter) was used fortransfection, no similar effect was observed.

An “antisense” MN cDNA/MN promoter construct was used to transfect CGL3cells. The effect was the opposite of that of the CGL1 cells transfectedwith the “sense” construct. Whereas the transfected CGL1 cells formedcolonies several times larger than the control CGL1, the transfectedCGL3 cells formed colonies much smaller than the control CGL3 cells.

For those experiments, the part of the promoter region that was linkedto the MN cDNA through BamHI site was derived from NcoI-BamHI fragmentof the MN genomic clone and represents the region 233 bp upstream fromthe transcription initiation site. After the ligation, the joint DNA wasinserted into a pBK-CMV expression vector [Stratagene]. The requiredorientation of the inserted sequence was ensured by directional cloningand subsequently verified by restriction analysis. The transfectionprocedure was the same as used in transfecting the NIH 3T3 cells(Example 15), but co-transfection with the pSV2neo plasmid was notnecessary since the neo selection marker was already included in thepBK-CMV vector.

After two weeks of selection in a medium containing G418, remarkabledifferences between the numbers and sizes of the colonies grown wereevident as noted above. Immediately following the selection and cloning,the MN-transfected CGL1 and CGL3 cells were tested by SP-RIA forexpression and repression of MN, respectively. The isolated transfectedCGL1 clones were MN positive (although the level was lower than obtainedwith the full-length cDNA), whereas MN protein was almost absent fromthe transfected CGL3 clones. However, in subsequent passages, theexpression of MN in transfected CGL1 cells started to cease, and wasthen blocked perhaps evidencing a control feedback mechanism.

As a result of the very much lowered proliferation of the transfectedCGL3 cells, it was difficult to expand the majority of cloned cells(according to SP-RIA, those with the lowest levels of MN), and they werelost during passaging. However, some clones overcame that problem andagain expressed MN. It is possible that once those cells reached ahigher quantity, that the level of endogenously produced MN mRNAincreased over the amount of ectopically expressed antisense mRNA.

Nucleic Acid Probes and Test Kits

Nucleic acid probes of this invention are those comprising sequencesthat are complementary or substantially complementary to the MN cDNAsequences shown in FIGS. 1A-1B and 15 or to other MN gene sequences,such as, the genomic clone sequence of FIG. 25 [SEQ. ID. NO.: 23]. Thephrase “substantially complementary” is defined herein to have themeaning as it is well understood in the art and, thus, used in thecontext of standard hybridization conditions. The stringency ofhybridization conditions can be adjusted to control the precision ofcomplementarity. Exemplary are the stringent hybridization conditionsused in Examples 11 and 12. Two nucleic acids are, for example,substantially complementary to each other, if they hybridize to eachother under such stringent hybridization conditions.

Stringent hybridization conditions are considered herein to conform tostandard hybridization conditions understood in the art to be stringent.For example, it is generally understood that stringent conditionsencompass relatively low salt and/or high temperature conditions, suchas provided by 0.02 M to 0.15 M NaCl at temperatures of 50° C. to 70° C.Less stringent conditions, such as, 0.15 M to 0.9 M salt at temperaturesranging from 20° C. to 55° C. can be made more stringent by addingincreasing amounts of formamide, which serves to destabilize hybridduplexes as does increased temperature.

Exemplary stringent hybridization conditions are described in Examples11 and 12, infra; the hybridizations therein were carried out “in thepresence of 50% formamide at 42° C.” [See Sambrook et al., MolecularCloning: A Laboratory Manual, pages 1.91 and 9.47-9.51 (Second Edition,Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989);Maniatis et al., Molecular Cloning: A Laboratory Manual, pages 387-389(Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y.; 1982);Tsuchiya et al., Oral Surgery, Oral Medicine, Oral Pathology, 71(6):721-725 (June 1991).]

Preferred nucleic acid probes of this invention are fragments of theisolated nucleic acid sequences that encode MN proteins or polypeptidesaccording to this invention. Preferably those probes are composed of atleast fifty nucleotides.

However, nucleic acid probes of this invention need not hybridize to acoding region of MN. For example, nucleic acid probes of this inventionmay hybridize partially or wholly to a non-coding region of the genomicclone of FIG. 25 a-b [SEQ. ID. NO.: 23]. Conventional technology can beused to determine whether fragments of SEQ. ID. NO.: 23 or relatednucleic acids are useful to identify MN nucleic acid sequences. [See,for example, Benton and Davis, supra and Fuscoe et al., supra.]

Nucleic acid probes of this invention can be used to detect MN DNAand/or RNA, and thus can be used to test for the presence or absence ofMN genes, and amplification(s), mutation(s) or genetic rearrangements ofMN genes in the cells of a patient. For example, overexpression of an MNgene may be detected by Northern blotting using probes of thisinvention. Gene alterations, as amplifications, translocations,inversions, and deletions among others, can be detected by using probesof this invention for in situ hybridization to chromosomes from apatient's cells, whether in metaphase spreads or interphase nuclei.Southern blotting could also be used with the probes of this inventionto detect amplifications or deletions of MN genes. Restriction FragmentLength Polymorphism (RFLP) analysis using said probes is a preferredmethod of detecting gene alterations, mutations and deletions. Saidprobes can also be used to identify MN proteins and/or polypeptides aswell as homologs or near homologs thereto by their hybridization tovarious mRNAs transcribed from MN genes in different tissues.

Probes of this invention thus can be usefuldiagnostically/prognostically. Said probes can be embodied in test kits,preferably with appropriate means to enable said probes when hybridizedto an appropriate MN gene or MN mRNA target to be visualized. Suchsamples include tissue specimens including smears, body fluids andtissue and cell extracts.

PCR Assays. To detect relatively large genetic rearrangements,hybridization tests can be used. To detect relatively small geneticrearrangements, as, for example, small deletions or amplifications, orpoint mutations, the polymerase chain reaction (PCR) would preferably beused. [U.S. Pat. Nos. 4,800,159; 4,683,195; 4,683,202; and Chapter 14 ofSambrook et al., Molecular Cloning: A Laboratory Manual, supra]

An exemplary assay would use cellular DNA from normal and cancerouscells, which DNA would be isolated and amplified employing appropriatePCR primers. The PCR products would be compared, preferably initially,on a sizing gel to detect size changes indicative of certain geneticrearrangements. If no differences in sizes are noted, furthercomparisons can be made, preferably using, for example,PCR-single-strand conformation polymorphism (PCR-SSCP) assay or adenaturing gradient gel electrophoretic assay. [See, for example,Hayashi, K., “PCR-SSCP: A Simple and Sensitive Method for Detection ofMutations in the Genomic DNA,” in PCR Methods and Applications, 1: 34-38(1991); and Meyers et al., “Detection and Localization of Single BaseChanges by Denaturing Gradient Gel Electrophoresis,” Methods inEnzymology, 155: 501 (1987).]

Assays

Assays according to this invention are provided to detect and/orquantitate MN antigen or MN-specific antibodies in vertebrate samples,preferably mammalian samples, more preferably human samples. Suchsamples include tissue specimens, body fluids, tissue extracts and cellextracts. MN antigen may be detected by immunoassay, immunohistochemicalstaining, immunoelectron and scanning microscopy using immunogold amongother techniques.

Preferred tissue specimens to assay by immunohistochemical staininginclude cell smears, histological sections from biopsied tissues ororgans, and imprint preparations among other tissue samples. Such tissuespecimens can be variously maintained, for example, they can be fresh,frozen, or formalin-, alcohol- or acetone- or otherwise fixed and/orparaffin-embedded and deparaffinized. Biopsied tissue samples can be,for example, those samples removed by aspiration, bite, brush, cone,chorionic villus, endoscopic, excisional, incisional, needle,percutaneous punch, and surface biopsies, among other biopsy techniques.

Preferred cervical tissue specimens include cervical smears, conizationspecimens, histologic sections from hysterectomy specimens or otherbiopsied cervical tissue samples. Preferred means of obtaining cervicalsmears include routine swab, scraping or cytobrush techniques, amongother means. More preferred are cytobrush or swab techniques.Preferably, cell smears are made on microscope slides, fixed, forexample, with 55% EtOH or an alcohol based spray fixative and air-dried.

Papanicolaou-stained cervical smears (Pap smears) can be screened by themethods of this invention, for example, for retrospective studies.Preferably, Pap smears would be decolorized and re-stained with labeledantibodies against MN antigen. Also archival specimens, for example,matched smears and biopsy and/or tumor specimens, can be used forretrospective studies. Prospective studies can also be done with matchedspecimens from patients that have a higher than normal risk ofexhibiting abnormal cervical cytopathology.

Preferred samples in which to assay MN antigen by, for example, Westernblotting or radioimmunoassay, are tissue and/or cell extracts. However,MN antigen may be detected in body fluids, which can include among otherfluids: blood, serum, plasma, semen, breast exudate, saliva, tears,sputum, mucous, urine, lymph, cytosols, ascites, pleural effusions,amniotic fluid, bladder washes, bronchioalveolar lavages andcerebrospinal fluid. It is preferred that the MN antigen be concentratedfrom a larger volume of body fluid before testing. Preferred body fluidsto assay would depend on the type of cancer for which one was testing,but in general preferred body fluids would be breast exudate, pleuraleffusions and ascites.

MN-specific antibodies can be bound by serologically active MNproteins/polypeptides in samples of such body fluids as blood, plasma,serum, lymph, mucous, tears, urine, spinal fluid and saliva; however,such antibodies are found most usually in blood, plasma and serum,preferably in serum. A representative assay to detect MN-specificantibodies is shown in Example 8 below wherein the fusion proteinGEX-3X-MN is used. Correlation of the results from the assays to detectand/or quantitate MN antigen and MN specific antibodies reactivetherewith, provides a preferred profile of the disease condition of apatient.

The assays of this invention are both diagnostic and/or prognostic,i.e., diagnostic/prognostic. The term “diagnostic/prognostic” is hereindefined to encompass the following processes either individually orcumulatively depending upon the clinical context: determining thepresence of disease, determining the nature of a disease, distinguishingone disease from another, forecasting as to the probable outcome of adisease state, determining the prospect as to recovery from a disease asindicated by the nature and symptoms of a case, monitoring the diseasestatus of a patient, monitoring a patient for recurrence of disease,and/or determining the preferred therapeutic regimen for a patient. Thediagnostic/prognostic methods of this invention are useful, for example,for screening populations for the presence of neoplastic orpre-neoplastic disease, determining the risk of developing neoplasticdisease, diagnosing the presence of neoplastic and/or pre-neoplasticdisease, monitoring the disease status of patients with neoplasticdisease, and/or determining the prognosis for the course of neoplasticdisease. For example, it appears that the intensity of theimmunostaining with MN-specific antibodies may correlate with theseverity of dysplasia present in samples tested.

The present invention is useful for screening for the presence of a widevariety of neoplastic diseases including carcinomas, such as, mammary,urinary tract, ovarian, uterine, cervical, endometrial, squamous celland adenosquamous carcinomas; head and neck cancers; mesodermal tumors,such as, neuroblastomas and retinoblastomas; sarcomas, such asosteosarcomas and Ewing's sarcoma; and melanomas. Of particular interestare gynecological cancers including ovarian, uterine, cervical, vaginal,vulval and endometrial cancers, particularly ovarian, uterine cervicaland endometrial cancers. Also of particular interest are cancers of thebreast, of the stomach including esophagus, of the colon, of the kidney,of the prostate, of the liver, of the urinary tract including bladder,of the lung, and of the head and neck.

The invention provides methods and compositions for evaluating theprobability of the presence of malignant or pre-malignant cells, forexample, in a group of cells freshly removed from a host. Such an assaycan be used to detect tumors, quantitate their growth, and help in thediagnosis and prognosis of disease. The assays can also be used todetect the presence of cancer metastasis, as well as confirm the absenceor removal of all tumor tissue following surgery, cancer chemotherapyand/or radiation therapy. It can further be used to monitor cancerchemotherapy and tumor reappearance.

The presence of MN antigen or antibodies can be detected and/orquantitated using a number of well-defined diagnostic assays. Those inthe art can adapt any of the conventional immunoassay formats to detectand/or quantitate MN antigen and/or antibodies. Example 8 details theformat of a preferred diagnostic method of this invention—aradioimmunoassay. Immunohistochemical staining is another preferredassay format as exemplified in Example 13.

Many other formats for detection of MN antigen and MN-specificantibodies are, of course available. Those can be Western blots, ELISAs(enzyme-linked immunosorbent assays), RIAs (radioimmunoassay),competitive EIA or dual antibody sandwich assays, among other assays allcommonly used in the diagnostic industry. In such immunoassays, theinterpretation of the results is based on the assumption that theantibody or antibody combination will not cross-react with otherproteins and protein fragments present in the sample that are unrelatedto MN.

Representative of one type of ELISA test for MN antigen is a formatwherein a microtiter plate is coated with antibodies made to MNproteins/polypeptides or antibodies made to whole cells expressing MNproteins, and to this is added a patient sample, for example, a tissueor cell extract. After a period of incubation permitting any antigen tobind to the antibodies, the plate is washed and another set of anti-MNantibodies which are linked to an enzyme is added, incubated to allowreaction to take place, and the plate is then rewashed. Thereafter,enzyme substrate is added to the microtiter plate and incubated for aperiod of time to allow the enzyme to work on the substrate, and theabsorbance of the final preparation is measured. A large change inabsorbance indicates a positive result.

It is also apparent to one skilled in the art of immunoassays that MNproteins and/or polypeptides can be used to detect and/or quantitate thepresence of MN antigen in the body fluids, tissues and/or cells ofpatients. In one such embodiment, a competition immunoassay is used,wherein the MN protein/polypeptide is labeled and a body fluid is addedto compete the binding of the labeled MN protein/polypeptide toantibodies specific to MN protein/polypeptide. Such an assay can be usedto detect and/or quantitate MN antigen as described in Example 8.

In another embodiment, an immunometric assay may be used wherein alabeled antibody made to a MN protein or polypeptide is used. In such anassay, the amount of labeled antibody which complexes with theantigen-bound antibody is directly proportional to the amount of MNantigen in the sample.

A representative assay to detect MN-specific antibodies is a competitionassay in which labeled MN protein/polypeptide is precipitated byantibodies in a sample, for example, in combination with monoclonalantibodies recognizing MN proteins/polypeptides. One skilled in the artcould adapt any of the conventional immunoassay formats to detect and/orquantitate MN-specific antibodies. Detection of the binding of saidantibodies to said MN protein/polypeptide could be by many ways known tothose in the art, e.g., in humans, with the use of anti-human labeledIgG.

An exemplary immunoassay method of this invention to detect and/orquantitate MN antigen in a vertebrate sample comprises the steps of:

a) incubating said vertebrate sample with one or more sets of antibodies(an antibody or antibodies) that bind to MN antigen wherein one set islabeled or otherwise detectable;

b) examining the incubated sample for the presence of immune complexescomprising MN antigen and said antibodies.

Another exemplary immunoassay method according to this invention is thatwherein a competition immunoassay is used to detect and/or quantitate MNantigen in a vertebrate sample and wherein said method comprises thesteps of:

a) incubating a vertebrate sample with one or more sets of MN-specificantibodies and a certain amount of a labeled or otherwise detectable MNprotein/polypeptide wherein said MN protein/polypeptide competes forbinding to said antibodies with MN antigen present in the sample;

b) examining the incubated sample to determine the amount oflabeled/detectable MN protein/polypeptide bound to said antibodies; and

c) determining from the results of the examination in step b) whether MNantigen is present in said sample and/or the amount of MN antigenpresent in said sample.

Once antibodies (including biologically active antibody fragments)having suitable specificity have been prepared, a wide variety ofimmunological assay methods are available for determining the formationof specific antibody-antigen complexes. Numerous competitive andnon-competitive protein binding assays have been described in thescientific and patent literature, and a large number of such assays arecommercially available. Exemplary immunoassays which are suitable fordetecting a serum antigen include those described in U.S. Pat. Nos.3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; and 4,098,876.

Antibodies employed in assays may be labeled or unlabeled. Unlabeledantibodies may be employed in agglutination; labeled antibodies may beemployed in a wide variety of assays, employing a wide variety oflabels.

Suitable detection means include the use of labels such asradionuclides, enzymes, coenzymes, fluorescers, chemiluminescers,chromogens, enzyme substrates or co-factors, enzyme inhibitors, freeradicals, particles, dyes and the like. Such labeled reagents may beused in a variety of well known assays, such as radioimmunoassays,enzyme immunoassays, e.g., ELISA, fluorescent immunoassays, and thelike. See for example, U.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837;and 4,233,402.

Methods to prepare antibodies useful in the assays of the invention aredescribed below. The examples below detail representative assaysaccording to this invention.

Immunoassay Test Kits

The above outlined assays can be embodied in test kits to detect and/orquantitate MN antigen and/or MN-specific antibodies (includingbiologically active antibody fragments). Kits to detect and/orquantitate MN antigen can comprise MN protein(s)/polypeptides(s) and/orMN-specific antibodies, polyclonal and/or monoclonal. Suchdiagnostic/prognostic test kits can comprise one or more sets ofantibodies, polyclonal and/or monoclonal, for a sandwich format whereinantibodies recognize epitopes on the MN antigen, and one set isappropriately labeled or is otherwise detectable.

Test kits for an assay format wherein there is competition between alabeled (or otherwise detectable) MN protein/polypeptide and MN antigenin the sample, for binding to an antibody, can comprise the combinationof the labeled protein/polypeptide and the antibody in amounts whichprovide for optimum sensitivity and accuracy.

Test kits for MN-specific antibodies preferably compriselabeled/detectable MN proteins(s) and/or polypeptides(s), and maycomprise other components as necessary, for example, to perform apreferred assay as outlined in Example 8 below, such as, controls,buffers, diluents and detergents. Such test kits can have otherappropriate formats for conventional assays.

A kit for use in an enzyme-immunoassay typically includes anenzyme-labelled reagent and a substrate for the enzyme. The enzyme can,for example, bind either an MN-specific antibody of this invention or toan antibody to such an MN-specific antibody.

Preparation of MN-Specific Antibodies

The term “antibodies” is defined herein to include not only wholeantibodies but also biologically active fragments of antibodies,preferably fragments containing the antigen binding regions. Suchantibodies may be prepared by conventional methodology and/or by geneticengineering. Antibody fragments may be genetically engineered,preferably from the variable regions of the light and/or heavy chains(V_(H) and V_(L)), including the hypervariable regions, and still morepreferably from both the V_(H) and V_(L) regions. For example, the term“antibodies” as used herein comprehends polyclonal and monoclonalantibodies and biologically active fragments thereof including amongother possibilities “univalent” antibodies [Glennie et al., Nature, 295:712 (1982)]; Fab proteins including Fab′ and F(ab′)₂ fragments whethercovalently or non-covalently aggregated; light or heavy chains alone,preferably variable heavy and light chain regions (V_(H) and V_(L)regions), and more preferably including the hypervariable regions[otherwise known as the complementarity determining regions (CDRs) ofsaid V_(H) and V_(L) regions]; F_(c) proteins; “hybrid” antibodiescapable of binding more than one antigen; constant-variable regionchimeras; “composite” immunoglobulins with heavy and light chains ofdifferent origins; “altered” antibodies with improved specificity andother characteristics as prepared by standard recombinant techniques andalso by oligonucleotide-directed mutagenesis techniques[Dalbadie-McFarland et al., PNAS (USA), 79: 6409 (1982)].

It may be preferred for therapeutic and/or imaging uses that theantibodies be biologically active antibody fragments, preferablygenetically engineered fragments, more preferably genetically engineeredfragments from the V_(H) and/or V_(L) regions, and still more preferablycomprising the hypervariable regions thereof.

There are conventional techniques for making polyclonal and monoclonalantibodies well-known in the immunoassay art. Immunogens to prepareMN-specific antibodies include MN proteins and/or polypeptides,preferably purified, and MX-infected tumor line cells, for example,MX-infected HeLa cells, among other immunogens.

Anti-peptide antibodies are also made by conventional methods in the artas described in European Patent Publication No. 44,710 (published Jan.27, 1982). Briefly, such anti-peptide antibodies are prepared byselecting a peptide from an MN amino acid sequence as from FIG. 1A-B or15, chemically synthesizing it, conjugating it to an appropriateimmunogenic protein and injecting it into an appropriate animal, usuallya rabbit or a mouse; then, either polyclonal or monoclonal antibodiesare made, the latter by a Kohler-Milstein procedure, for example.

Besides conventional hybridoma technology, newer technologies can beused to produce antibodies according to this invention. For example, theuse of the PCR to clone and express antibody V-genes and phage displaytechnology to select antibody genes encoding fragments with bindingactivities has resulted in the isolation of antibody fragments fromrepertoires of PCR amplified V-genes using immunized mice or humans.[Marks et al., BioTechnology, 10: 779 (July 1992) for references; Chianget al., BioTechniques, 7(4): 360 (1989); Ward et al., Nature, 341: 544(Oct. 12, 1989); Marks et al., J. Mol. Biol., 222: 581 (1991); Clacksonet al., Nature, 352: (15 Aug. 1991); and Mullinax et al., PNAS (USA),87: 8095 (October 1990).]

Descriptions of preparing antibodies, which term is herein defined toinclude biologically active antibody fragments, by recombinanttechniques can be found in U.S. Pat. No. 4,816,567 (issued Mar. 28,1989); European Patent Application Publication Number (EP) 338,745(published Oct. 25, 1989); EP 368,684 (published Jun. 16, 1990); EP239,400 (published Sep. 30, 1987); WO 90/14424 (published Nov. 29,1990); WO 90/14430 (published May 16, 1990); Huse et al., Science, 246:1275 (Dec. 8, 1989); Marks et al., BioTechnology, 10: 779 (July 1992);La Sastry et al., PNAS (USA), 86: 5728 (August 1989); Chiang et al.,BioTechniques, 7(40): 360 (1989); Orlandi et al., PNAS (USA), 86: 3833(May 1989); Ward et al. Nature, 341: 544 (Oct. 12, 1989); Marks et al.,J. Mol. Biol., 222: 581 (1991); and Hoogenboom et al., Nucleic AcidsRes., 19(15): 4133 (1991).

Representative Mabs

Monoclonal antibodies for use in the assays of this invention may beobtained by methods well known in the art for example, Galfre andMilstein, “Preparation of Monoclonal Antibodies: Strategies andProcedures,” in Methods in Enzymology: Immunochemical Techniques. 73:1-46 [Langone and Vanatis (eds); Academic Press (1981)]; and in theclassic reference, Milstein and Kohler, Nature, 256: 495-497 (1975).]

Although representative hybridomas of this invention are formed by thefusion of murine cell lines, human/human hybridomas [Olsson et al., PNAS(USA), 77: 5429 (1980)] and human/murine hybridomas [Schlom et al., PNAS(USA), 77: 6841 (1980); Shearman et al. J. Immunol., 146: 928-935(1991); and Gorman et al., PNAS (USA), 88: 4181-4185 (1991)] can also beprepared among other possiblities. Such humanized monoclonal antibodieswould be preferred monoclonal antibodies for therapeutic and imaginguses.

Monoclonal antibodies specific for this invention can be prepared byimmunizing appropriate mammals, preferably rodents, more preferablyrabbits or mice, with an appropriate immunogen, for example,MaTu-infected HeLa cells, MN fusion proteins, or MNproteins/polypeptides attached to a carrier protein if necessary.Exemplary methods of producing antibodies of this invention aredescribed below.

The monoclonal antibodies useful according to this invention to identifyMN proteins/polypeptides can be labeled in any conventional manner, forexample, with enzymes such as horseradish peroxidase (HRP), fluorescentcompounds, or with radioactive isotopes such as, ¹²⁵I, among otherlabels. A preferred label, according to this invention is ¹²⁵I, and apreferred method of labeling the antibodies is by using chloramine-T[Hunter, W. M., “Radioimmunoassay,” In: Handbook of ExperimentalImmunology, pp. 14.1-14.40 (D. W. Weir ed.; Blackwell,Oxford/London/Edinburgh/Melbourne; 1978)].

Representative mabs of this invention include Mabs M75, MN9, MN12 andMN7 described below. Monoclonal antibodies of this invention serve toidentify MN proteins/polypeptides in various laboratory diagnostictests, for example, in tumor cell cultures or in clinical samples.

Mabs Prepared Against HeLa Cells

MAb M75. Monoclonal antibody M75 (MAb M75) is produced by mouselymphocytic hybridoma VU-M75, which was initially deposited in theCollection of Hybridomas at the Institute of Virology, Slovak Academy ofSciences (Bratislava, Slovakia) and was deposited under ATCC DesignationHB 11128 on Sep. 17, 1992 at the American Type Culture Collection (ATCC)in Manassas, Va. (USA).

Hybridoma VU-M75 was produced according to the procedure described inGerhard, W., “Fusion of cells in suspension and outgrowth of hybrids inconditioned medium,” In: Monoclonal Antibodies. Hebridomas: A NewDimension in Biological Analysis, page 370 [Kennet et al. (eds.); PlenumN.Y. (USA)]. BALB/C mice were immunized with MaTu-infected HeLa cells,and their spleen cells were fused with myeloma cell line NS-0. Tissueculture media from the hybridomas were screened for monoclonalantibodies, using as antigen the p58 immunoprecipitated from cellextracts of MaTu-infected HeLa with rabbit anti-MaTu serum and proteinA-Staphylococcus aureus cells (SAC) [Zavada and Zavadova, Arch. Virol.,118 189-197 (1991)], and eluted from SDS-PAGE gels. Monoclonalantibodies were purified from TC media by affinity chromatography onprotein A-Sepharose [Harlow and Lane, “Antibodies: A Laboratory Manual,”Cold Spring Harbor, Cold Spring Harbor, N.Y. (USA); 1988].

Mab M75 recognizes both the nonglycosylated GEX-3X-MN fusion protein andnative MN protein as expressed in CGL3 cells equally well. Mab M75 wasshown by epitope mapping to be reactive with the epitope represented bythe amino acid sequence from AA 62 to AA 67 [SEQ. ID. NO.: 10] of the MNprotein shown in FIG. 15.

Mabs M16 and M67. Also produced by the method described for producingMAb M75 (isotype IgG2B) were MAbs M16 (isotype IgG2A) and M67 (isotypeIgG1). Mabs M16 and M67 recognize MX protein, as described in theexamples below.

MAb H460. Monoclonal antibody H460 (MAb H460) was prepared in a mannersimilar to that for MAb M75 except that the mice were immunized withHeLa cells uninfected with MaTu, and lymphocytes of the mice rather thanspleen cells were fused with cells from myeloma cell line NS-0. MAb H460reacts about equally with any human cells.

Mabs Prepared Against Fusion Protein GEX-3X-MN

Monoclonal antibodies of this invention were also prepared against theMN glutathione S-transferase fusion protein (GEX-3X-MN) purified byaffinity chromatography as described above. BALB/C mice were immunizedintraperitoneally according to standard procedures with the GEX-3X-MNfusion protein in Freund's adjuvant. Spleen cells of the mice were fusedwith SP/20 myeloma cells [Milstein and Kohler, supra].

Tissue culture media from the hybridomas were screened against CGL3 andCGL1 membrane extracts in an ELISA employing HRP labelled-rabbitanti-mouse. The membrane extracts were coated onto microtiter plates.Selected were antibodies reacted with the CGL3 membrane extract.Selected hybridomas were cloned twice by limiting dilution.

The mabs prepared by the just described method were characterized byWestern blots of the GEX-3X-MN fusion protein, and with membraneextracts from the CGL1 and CGL3 cells. Representative of the mabsprepared are Mabs MN9, MN12 and MN7.

Mab MN9. Monoclonal antibody MN9 (Mab MN9) reacts to the same epitope asMab M75, represented by the sequence from AA 62 to AA 67 [SEQ. ID. NO.:10] of the FIG. 15 MN protein. As Mab M75, Mab MN9 recognizes both theGEX-3X-MN fusion protein and native MN protein equally well.

Mabs corresponding to Mab MN9 can be prepared reproducibly by screeninga series of mabs prepared against an MN protein/polypeptide, such as,the GEX-3X-MN fusion protein, against the peptide representing theepitope for Mabs M75 and MN9. That peptide is Arg Arg Ile Cys Pro Val[SEQ. ID. NO.: 10]. Alternatively, the Novatope system [Novagen] orcompetition with the deposited Mab M75 could be used to select mabscomparable to Mabs M75 and MN9.

Mab MN12. Monoclonal antibody MN12 (Mab MN12) is produced by the mouselymphocytic hybridoma MN 12.2.2 which was deposited under ATCCDesignation HB 11647 on Jun. 9, 1994 at the American Type CultureCollection (ATCC) at 10801 University Blvd., Manassas, Va. 20110-2209(USA). Antibodies corresponding to Mab MN12 can also be made,analogously to the method outlined above for Mab MN9, by screening aseries of antibodies prepared against an MN protein/polypeptide, againstthe peptide representing the epitope for Mab MN12. That peptide is GlyLys Met Thr His Trp [SEQ. ID. NO.: 12]. The Novatope system could alsobe used to find antibodies specific for said epitope.

Mab MN7. Monoclonal antibody MN7 (Mab MN7) was selected from mabsprepared against nonglycosylated GEX-3X-MN as described above. Itrecognizes the epitope on MN represented by the amino acid sequence fromAA 127 to AA 147 [SEQ. ID. NO.: 12; Asn Asn Ala His Arg Asp Lys Glu GlyAsp Asp Gln Ser His Trp Arg Tyr Gly Gly Asp Pro] of the FIG. 15 MNprotein. Analogously to methods described above for Mabs MN9 and MN12,mabs corresponding to Mab MN7 can be prepared by selecting mabs preparedagainst an MN protein/polypeptide that are reactive with the peptidehaving SEQ. ID. NO.: 12, or by the stated alternative means.

Epitope Mapping

Epitope mapping was performed by the Novatope system, a kit for which iscommercially available from Novagen, Inc. [See, for analogous example,Li et al., Nature, 363: 85-88 (6 May 1993).] In brief, the MN cDNA wascut into overlapping short fragments of approximately 60 base pairs. Thefragments were expressed in E. coli, and the E. coli colonies weretransferred onto nitrocellulose paper, lysed and probed with the mab ofinterest. The MN cDNA of clones reactive with the mab of interest wassequenced, and the epitopes of the mabs were deduced from theoverlapping polypeptides found to be reactive with each mab.

Therapeutic Use of MN-Specific Antibodies

The MN-specific antibodies of this invention, monoclonal and/orpolyclonal, preferably monoclonal, and as outlined above, may be usedtherapeutically in the treatment of neoplastic and/or pre-neoplasticdisease, either alone or in combination with chemotherapeutic drugs ortoxic agents, such as ricin A. Further preferred for therapeutic usewould be biologically active antibody fragments as described herein.Also preferred MN-specific antibodies for such therapeutic uses would behumanized monoclonal antibodies.

The MN-specific antibodies can be administered in a therapeuticallyeffective amount, preferably dispersed in a physiologically acceptable,nontoxic liquid vehicle.

Imaging Use of Antibodies

Further, the MN-specific antibodies of this invention when linked to animaging agent, such as a radionuclide, can be used for imaging.Biologically active antibody fragments or humanized monoclonalantibodies, may be preferred for imaging use.

A patient's neoplastic tissue can be identified as, for example, sitesof transformed stem cells, of tumors and locations of any metastases.Antibodies, appropriately labeled or linked to an imaging agent, can beinjected in a physiologically acceptable carrier into a patient, and thebinding of the antibodies can be detected by a method appropriate to thelabel or imaging agent, for example, by scintigraphy.

Antisense MN Nucleic Acid Sequences

MN genes are herein considered putative oncogenes and the encodedproteins thereby are considered to be putative oncoproteins. Antisensenucleic acid sequences substantially complementary to mRNA transcribedfrom MN genes, as represented by the antisense oligodeoxynucleotides(ODNs) of Example 10, infra, can be used to reduce or prevent expressionof the MN gene. [Zamecnick, P. C., “Introduction: Oligonucleotide BaseHybridization as a Modulator of Genetic Message Readout,” pp. 1-6,Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS,(Wiley-Liss, Inc., New York, N.Y., USA; 1991); Wickstrom, E., “AntisenseDNA Treatment of HL-60 Promyelocytic Leukemia Cells: TerminalDifferentiation and Dependence on Target Sequence,” pp. 7-24, id.;Leserman et al., “Targeting and Intracellular Delivery of AntisenseOligonucleotides Interfering with Oncogene Expression,” pp. 25-34, id.;Yokoyama, K., “Transcriptional Regulation of c-myc Proto-oncogene byAntisense RNA,” pp. 35-52, id.; van den Berg et al., “Antisense fosOligodeoxyribonucleotides Suppress the Generation of ChromosomalAberrations,” pp. 63-70, id.; Mercola, D., “Antisense fos and fun RNA,”pp. 83-114, id.; Inouye, Gene, 72: 25-34 (1988); Miller and Ts'o, Ann.Reports Med. Chem., 23: 295-304 (1988); Stein and Cohen, Cancer Res.,48: 2659-2668 (1988); Stevenson and Inversen, J. Gen. Virol., 70:2673-2682 (1989); Goodchild, “Inhibition of Gene Expression byOligonucleotides,” pp. 53-77, Oligodeoxynucleotides: AntisenseInhibitors of Gene Expression (Cohen, J. S., ed; CRC Press, Boca Raton,Fla., USA; 1989); Dervan et al., “Oligonucleotide Recognition ofDouble-helical DNA by Triple-helix Formation,” pp. 197-210, id.;Neckers, L. M., “Antisense Oligodeoxynucleotides as a Tool for StudyingCell Regulation: Mechanisms of Uptake and Application to the Study ofOncogene Function,” pp. 211-232, id.; Leitner et al., PNAS (USA), 87:3430-3434 (1990); Bevilacqua et al., PNAS (USA), 85: 831-835 (1988);Loke et al. Curr. Top. Microbiol. Immunol., 141: 282-288 (1988); Sarinet al., PNAS (USA), 85: 7448-7451 (1988); Agrawal et al., “AntisenseOligonucleotides: A Possible Approach for Chemotherapy and AIDS,”International Union of Biochemistry Conference on Nucleic AcidTherapeutics (Jan. 13-17, 1991; Clearwater Beach, Fla., USA); Armstrong,L., Ber. Week, pp. 88-89 (Mar. 5, 1990); and Weintraub et al., Trends,1: 22-25 (1985).] Such antisense nucleic acid sequences, preferablyoligonucleotides, by hybridizing to the MN mRNA, particularly in thevicinity of the ribosome binding site and translation initiation point,inhibits translation of the mRNA. Thus, the use of such antisensenucleic acid sequences may be considered to be a form of cancer therapy.

Preferred antisense oligonucleotides according to this invention aregene-specific ODNs or oligonucleotides complementary to the 5′ end of MNmRNA. Particularly preferred are the 29-mer ODN1 and 19-mer ODN2 forwhich the sequences are provided in Example 10, infra. Those antisenseODNs are representative of the many antisense nucleic acid sequencesthat can function to inhibit MN gene expression. Ones of ordinary skillin the art could determine appropriate antisense nucleic acid sequences,preferably antisense oligonucleotides, from the nucleic acid sequencesof FIGS. 1A-1B, 15 and 25 a-b.

Also, as described above, CGL3 cells transfected with an “antisense” MNcDNA/promoter construct formed colonies much smaller than control CGL3cells.

Vaccines

It will be readily appreciated that MN proteins and polypeptides of thisinvention can be incorporated into vaccines capable of inducingprotective immunity against neoplastic disease and a dampening effectupon tumorigenic activity. Efficacy of a representative MN fusionprotein GEX-3X-MN as a vaccine in a rat model is shown in Example 14.

MN proteins and/or polypeptides may be synthesized or preparedrecombinantly or otherwise biologically, to comprise one or more aminoacid sequences corresponding to one or more epitopes of the MN proteinseither in monomeric or multimeric form. Those proteins and/orpolypeptides may then be incorporated into vaccines capable of inducingprotective immunity. Techniques for enhancing the antigenicity of suchpolypeptides include incorporation into a multimeric structure, bindingto a highly immunogenic protein carrier, for example, keyhole limpethemocyanin (KLH), or diphtheria toxoid, and administration incombination with adjuvants or any other enhancers of immune response.

Preferred MN proteins/polypeptides to be used in a vaccine according tothis invention would be genetically engineered MN proteins. Preferredrecombinant MN protein are the GEX-3X-MN and MN 20-19 proteins.

A preferred exemplary use of such a vaccine of this invention would beits administration to patients whose MN-carrying-primary cancer had beensurgically removed. The vaccine may induce active immunity in thepatients and prevent recidivism or metastasis.

It will further be appreciated that anti-idiotype antibodies toantibodies to MN proteins/polypeptides are also useful as vaccines andcan be similarly formulated.

An amino acid sequence corresponding to an epitope of an MNprotein/polypeptide either in monomeric or multimeric form may also beobtained by chemical synthetic means or by purification from biologicalsources including genetically modified microorganisms or their culturemedia. [See Lerner, “Synthetic Vaccines”, Sci. Am. 248(2): 66-74(1983).] The protein/polypeptide may be combined in an amino acidsequence with other proteins/polypeptides including fragments of otherproteins, as for example, when synthesized as a fusion protein, orlinked to other antigenic or non-antigenic polypeptides of synthetic orbiological origin. In some instances, it may be desirable to fuse a MNprotein or polypeptide to an immunogenic and/or antigenic protein orpolypeptide, for example, to stimulate efficacy of a MN-based vaccine.

The term “corresponding to an epitope of an MN protein/polypeptide” willbe understood to include the practical possibility that, in someinstances, amino acid sequence variations of a naturally occurringprotein or polypeptide may be antigenic and confer protective immunityagainst neoplastic disease and/or anti-tumorigenic effects. Possiblesequence variations include, without limitation, amino acidsubstitutions, extensions, deletions, truncations, interpolations andcombinations thereof. Such variations fall within the contemplated scopeof the invention provided the protein or polypeptide containing them isimmunogenic and antibodies elicited by such a polypeptide or proteincross-react with naturally occurring MN proteins and polypeptides to asufficient extent to provide protective immunity and/or anti-tumorigenicactivity when administered as a vaccine.

Such vaccine compositions will be combined with a physiologicallyacceptable medium, including immunologically acceptable diluents andcarriers as well as commonly employed adjuvants such as Freund'sComplete Adjuvant, saponin, alum, and the like. Administration would bein immunologically effective amounts of the MN proteins or polypeptides,preferably in quantities providing unit doses of from 0.01 to 10.0micrograms of immunologically active MN protein and/or polypeptide perkilogram of the recipient's body weight. Total protective doses mayrange from 0.1 to about 100 micrograms of antigen.

Routes of administration, antigen dose, number and frequency ofinjections are all matters of optimization within the scope of theordinary skill in the art.

The following examples are for purposes of illustration only and notmeant to limit the invention in any way.

Materials and Methods

The following materials and methods were used in examples below.

MaTu-Infected and Uninfected HeLa Cells

MaTu agent [Zavada et al., Nature New Biol., 240: 124-125 (1972); Zavadaet al., J. Gen. Virol, 24: 327-337 (1974)] was from original “MaTu”cells [Widmaier et al., Arch. Geschwulstforsch, 44: 1-10 (1974)]transferred into our stock of HeLa by cocultivation with MaTu cellstreated with mitomycin C, to ensure that control and MaTu-infected cellswere comparable. MaTu cells were incubated for 3 hours at 37° C. inmedia with 5 μg/ml of mitomycin C [Calbiochem; LaJolla, Calif. (USA)].Mixed cultures were set to 2×10⁵ of mitomycin C-treated cells and 4×10⁵of fresh recipient cells in 5 ml of medium. After 3 days they were firstsubcultured and further passaged 1-2 times weekly.

Control HeLa cells were the same as those described in Zavada et al.(1972), supra.

Sera

Human sera from cancer patients, from patients suffering with variousnon-tumor complaints and from healthy women were obtained from theClinics of Obstetrics and Gynaecology at the Postgraduate MedicalSchool, Bratislava, Slovakia. Human serum KH was from a fifty year oldmammary carcinoma patient, fourteen months after resection. That serumwas one of two sera out of 401 serum samples that contained neutralizingantibodies to the VSV(MaTU) pseudotype as described in Zavada et al.(1972), supra. Serum L8 was from a patient with Paget's disease. SerumM7 was from a healthy donor.

Rabbit anti-MaTu serum was prepared by immunizing a rabbit three timesat intervals of 30 days with 1-5×10⁷ viable MaTu-infected HeLa cells.

RIP and PAGE

RIP and PAGE were performed essentially as described in Zavada andZavadova, supra, except that in the experiments described herein[³⁵S]methionine (NEN), 10 μCi/ml of methionine-free MEM medium,supplemented with 2% FCS and 3% complete MEM were used. Confluent petridish cultures of cells were incubated overnight in that media.

For RIP, the SAC procedure [Kessler, J. Immunol., 115: 1617-1624 (1975)]was used. All incubations and centrifugations were performed at 0-4° C.Cell monolayers were extracted with RIPA buffer (0.14 M NaCl, 7.5 mMphosphate buffer, pH 7.2, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM phenylmethylsulfonyl fluoride and Trasylol). To reduce non-specificreactions, antisera were preabsorbed with fetal calf serum [Barbacid etal., PNAS (USA), 77: 1617-1621 (1980)] and antigenic extracts with SAC.

For PAGE (under reducing conditions) we used 10% gels with SDS [Laemmli,Nature, 227: 680-685 (1970)]. As reference marker proteins served theSigma kit [product MW-SDS-200; St. Louis, Mo. (USA)]. For fluorographywe used salicylate [Heegaard et al., Electrophoresis, 5: 263-269(1984)].

Immunoblots

Immunoblotting used as described herein follows the method of Towbin etal., PNAS (USA), 76: 4350-4354 (1979). The proteins were transferredfrom the gels onto nitrocellulose [Schleicher and Schuell; DasselGermany; 0.45 μm porosity] in Laemmli electrode buffer diluted 1:10 withdistilled water, with no methanol or SDS. The transfer was for 2½ hoursat 1.75 mA/cm². The blots were developed with ¹²⁵I-labeled MAbs andautoradiography was performed using intensifying screens, with X-rayfilms exposed at −70° C.

In extracts from cell cultures containing only small amounts of MNantigen, we concentrated the antigen from 0.5 or 1 ml of an extract byadding 50 μl of a 10% SAC suspension, pre-loaded with MAb M75. Thismethod allowed the concentration of MN antigen even from clinicalspecimens, containing human IgG; preliminary control experiments showedthat such a method did not interfere with the binding of the MN antigento SAC-adsorbed M75. Tissue extracts were made by grinding the tissuewith a mortar and pestle and sand (analytical grade). To the homogenateswas added RIPA buffer, 10:1 (volume to weight) of original tissue. Theextracts were clarified for 3 minutes on an Eppendorf centrifuge.

Example 1 Immunofluorescence of MaTu-Specific Antigens

Immunofluorescence experiments were performed on control andMaTu-infected HeLa cells with monoclonal antibodies, prepared asdescribed above, which are specific for MaTu-related antigens.FITC-conjugated anti-mouse IgG was used to detect the presence of themonoclonal antibodies. Staining of the cells with Giemsa revealed noclear differences between control and MaTu-infected HeLa cells.

MAbs, which in preliminary tests proved to be specific for MaTu-relatedantigens, showed two different reactivities in immunofluorescence. Arepresentative of the first group, MAb M67, gave a granular cytoplasmicfluorescence in MaTu-infected HeLa, which was only seen in cells fixedwith acetone; living cells showed no fluorescence. MAb M16 gave the sametype of fluorescence. With either M67 or M16, only extremely weak“background” fluorescence was seen in control HeLa cells.

Another MAb, M75, showed a granular membrane fluorescence on livingMaTu-infected cells and a granular nuclear fluorescence in acetone-fixedcells. However, M75 sometimes showed a similar, although much weaker,fluorescence on uninfected HeLa cells. A relationship was observed basedupon the conditions of growth: in HeLa cells uninfected with MaTu, bothtypes of fluorescence with MAb M75 were observed only if the cells weregrown for several passages in dense cultures, but not in sparse ones.

The amount of M75-reactive cell surface antigen was analyzedcytofluorometrically and was dependent on the density of the cellcultures and on infection with MaTu. Control and MaTu infected HeLacells were grown for 12 days in dense or sparse cultures. The cells werereleased with Versene (EDTA), and incubated with MAb M75 or with no MAb,and subsequently incubated with FITC-conjugated anti-mouse IgG. Theintensity of fluorescence was measured.

It appeared that the antigen binding MAb M75 is inducible: it was foundto be absent in control HeLa grown in sparse culture, and to be inducedeither by the growth of HeLa in dense culture or by infection with MaTu.Those two factors were found to have an additive or synergistic effect.Those observations indicated along with other results described hereinthat there were two different agents involved: exogenous, transmissibleMX, reactive with M67, and endogenous, inducible MN, detected by MAbM75.

Example 2 Immunoblot Analysis of Protein(s) Reactive with MAb M75

To determine whether MAb M75 reacts with the same protein in bothuninfected and MaTu-infected HeLa, and to determine the molecular weightof the protein, extracts of those cells were analyzed by PAGE andimmunoblotting (as described above). HeLa cells uninfected orMaTu-infected, that had been grown for 12 days in dense or sparsecultures, were seeded in 5-cm petri dishes, all variants at 5×10⁵ cellsper dish. Two days later, the cells were extracted with RIPA buffer(above described), 200 μl/dish. The extracts were mixed with 2×concentrated Laemmli sample buffer containing 6% mercaptoethanol andboiled for five minutes. Proteins were separated by SDS-PAGE and blottedon nitrocellulose. The blots were developed with ¹²⁵I-labeled MAb M75and autoradiography.

MAb M75 reacted with two MN-specific protein bands of 54 kd and 58 kd,which were the same in uninfected HeLa grown at high density and inMaTu-infected HeLa, evidencing that M75 recognizes the same protein(s)in both uninfected and MaTu-infected HeLa cells. Consistent with thecytofluorometric results, the amount of the antigen depended both oncell density and on infection with MaTu, the latter being a much morepotent inducer of p54/58N.

Example 3 Radioimmunoassay of MaTu-Specific Antigens In Situ

In contrast to the results with M75, the other MAb, M67, appeared to bespecific for the exogenous, transmissible agent MX. With M67 we observedno immunofluorescence in control HeLa, regardless of whether the cellswere grown in dense or in sparse culture. That difference was clearlyevidenced in radioimmunoassay experiments wherein ¹²⁵I-labeled MAbs M67and M75 were used.

For such experiments, parallel cultures of uninfected and MaTu-infectedcells were grown in dense or sparse cultures. The cultures were eitherlive (without fixation), or fixed (with methanol for five minutes andair-dried). The cultures were incubated for two hours in petri disheswith the ¹²⁵I-labeled MAbs, 6×10⁴ cpm/dish. Afterward, the cultures wererinsed four times with PBS and solubilized with 1 ml/dish of 2 N NaOH,and the radioactivity was determined on a gamma counter.

The simple radioimmunoassay procedure of this example was performeddirectly in petri dish cultures. Sixteen variants of theradioimmunoassay enabled us to determine whether the MX and MN antigensare located on the surface or in the interior of the cells and how theexpression of those two antigens depends on infection with MaTu and onthe density, in which the cells had been grown before the petri disheswere seeded. In live, unfixed cells only cell surface antigens can bindthe MAbs. In those cells, M67 showed no reaction with any variant of thecultures, whereas M75 reacted in accord with the results of Examples 1and 2 above.

Fixation of the cells with methanol made the cell membrane permeable tothe MAbs: M67 reacted with HeLa infected with MaTu, independently ofprevious cell density, and it did not bind to control HeLa. MAb M75 inmethanol-fixed cells confirmed the absence of corresponding antigen inuninfected HeLa from sparse cultures and its induction both by growth indense cultures and by infection with MaTu.

Example 4 Identification of MaTu Components Reactive with Animal Sera orAssociated with VSV Virions

Immunoblot analyses of MaTu-specific proteins from RIPA extracts fromuninfected or MaTu-infected HeLa and from purified VSV reproduced incontrol or in MaTu-infected HeLa, identified which of the antigens, p58Xor p54/58N, were radioimmunoprecipitated with animal sera, and which ofthem was responsible for complementation of VSV mutants and for theformation of pseudotype virions. Details concerning the procedures canbe found in Pastorekova et al., Virology, 187: 620-626 (1992).

The serum of a rabbit immunized with MaTu-infected HeLaimmunoprecipitated both MAb M67- and MAb M75-reactive proteins (bothp58X and p54/58N), whereas the “spontaneously” immune sera of normalrabbit, sheep or leukemic cow immunoprecipitated only the M67-reactiveprotein (p58X). On the other hand, in VSV reproduced in MaTu-infectedHeLa cells and subsequently purified, only the M75-reactive bands ofp54/58N were present. Thus, it was concluded that MX and MN areindependent components of MaTu, and that it was p54/58N thatcomplemented VSV mutants and was assembled into pseudotype virions.

As shown in FIG. 6 discussed below in Example 5, MX antigen was found tobe present in MaTu-infected fibroblasts. In Zavada and Zavadova, supra,it was reported that a p58 band from MX-infected fibroblasts could notbe detected by RIP with rabbit anti-MaTu serum. That serum contains moreantibodies to MX than to MN antigen. The discrepancy can be explained bythe extremely slow spread of MX in infected cultures. The resultsreported in Zavada and Zavadova, supra were from fibroblasts tested 6weeks after infection, whereas the later testing was 4 months afterinfection. We have found by immunoblots that MX can be first detected inboth H/F-N and H/F-T hybrids after 4 weeks, in HeLa cells after sixweeks and in fibroblasts only 10 weeks after infection.

Example 5 Expression of MN- and MX-Specific Proteins

FIG. 6 graphically illustrates the expression of MN- and MX-specificproteins in human fibroblasts, in HeLa cells and in H/F-N and H/F-Thybrid cells, and contrasts the expression in MX-infected and uninfectedcells. Cells were infected with MX by co-cultivation with mitomycinC-treated MX-infected HeLa. The infected and uninfected cells were grownfor three passages in dense cultures. About four months after infection,the infected cells concurrently with uninfected cells were grown inpetri dishes to produce dense monolayers.

A radioimmunoassay was performed directly in confluent petri dish (5 cm)culture of cells, fixed with methanol essentially as described inExample 3, supra. The monolayers were fixed with methanol and treatedwith ¹²⁵I-labeled MAbs M67 (specific for exogenous MX antigen) or M75(specific for endogenous MN antigen) at 6×10⁴ cpm/dish. The boundradioactivity was measured; the results are shown in FIG. 6.

FIG. 6 shows that MX was transmitted to all four cell lines tested, thatis, to human embryo fibroblasts, to HeLa and to both H/F-N and H/F-Thybrids; at the same time, all four uninfected counterpart cell lineswere MX-negative (top graph of FIG. 6). MN antigens are shown to bepresent in both MX-infected and uninfected HeLa and H/F-T cells, but notin the fibroblasts (bottom graph of FIG. 6). No MN antigen was found inthe control H/F-N, and only a minimum increase over background of MNantigen was found in MaTu infected H/F-N. Thus, it was found that in thehybrids, expression of MN antigen very strongly correlates withtumorigenicity.

Those results were consistent with the results obtained byimmunoblotting as shown in FIG. 7. The MN-specific twin protein p54/58Nwas detected in HeLa cell lines (both our standard type, that is, HeLaK, and in the Stanbridge mutant HeLa, that is, D98/AH.2 shown as HeLa S)and in tumorigenic H/F-T; however, p54/58N was not detected in thefibroblasts nor in the non-tumorigenic H/F-N even upon deliberately longexposure of the film used to detect radioactivity. Infection of the HeLacells with MX resulted in a strong increase in the concentration of thep54/58N protein(s).

The hybrid cells H/F-N and H/F-T were constructed by Eric J. Stanbridge[Stanbridge et al., Somatic Cell Genetics, 7: 699-712 (1981); andStanbridge et al., Science, 215: 252-259 (1982)]. His original hybrid,produced by the fusion of a HeLa cell and a human fibroblast was nottumorigenic in nude mice, although it retained some properties oftransformed cells, for example, its growth on soft agar. Rare segregantsfrom the hybrid which have lost chromosome 11 are tumorigenic. The mostlikely explanation for the tumorigenicity of those segregants is thatchromosome 11 contains a suppressor gene (an antioncogene), which blocksthe expression of a as yet unknown oncogene. The oncoprotein encoded bythat oncogene is critical for the capacity of the H/F hybrids to producetumors in nude mice. Since the p54/58N protein shows a correlation withthe tumorigenicity of H/F hybrids, it is a candidate for that putativeoncoprotein.

Example 6 Immunoblots of MN Antigen from Human Tumor Cell Cultures andfrom Clinical Specimens of Human Tissues

The association of MN antigen with tumorigenicity in the H/F hybridcells as illustrated by Example 5 prompted testing for the presence ofMN antigen in other human tumor cell cultures and in clinical specimens.Preliminary experiments indicated that the concentration of MN antigenin the extracts from other human tumor cell cultures was lower than inHeLa; thus, it was realized that long exposure of the autoradiographswould be required. Therefore, the sensitivity of the method wasincreased by the method indicated under Materials and Methods:Immunoblotting, supra, wherein the MN antigen was concentrated byprecipitation with MAb M75-loaded SAC.

FIG. 8 shows the immunoblots wherein lane A, a cell culture extract fromMX-infected HeLa cells was analysed directly (10 μl per lane) whereasthe antigens from the other extracts (lanes B-E) were each concentratedfrom a 500 μl extract by precipitation with MAb M75 and SAC.

FIG. 8 indicates that two other human carcinoma cell lines containMN-related proteins—T24 (bladder carcinoma; lane C) and T47D (mammarycarcinoma; lane D). Those cells contain proteins which react with MAbM75 that under reducing conditions have molecular weights of 54 kd and56 kd, and under non-reducing conditions have a molecular weight ofabout 153 kd. The intensity of those bands is at least ten times lowerthan that for the p54/58N twin protein from HeLa cells.

An extremely weak band at approximately 52 kd could be seen underreducing conditions from extracts from human melanoma cells (SK-Mel1477; lane E), but no bands for human fibroblast extracts (lane B) couldbe seen either on the reducing or non-reducing gels.

FIG. 9 shows immunoblots of human tissue extracts including surgicalspecimens as compared to a cell extract from MX-infected HeLa (lane A).The MN-related antigen from all the extracts but for lane A (analyseddirectly at 10 μl per lane) was first concentrated from a 1 ml extractas explained above. MN proteins were found in endometrial (lanes D andM), ovarian (lanes E and N) and in uterine cervical (lane 0) carcinomas.In those extracts MN-related proteins were found in three bands havingmolecular weights between about 48 kd and about 58 kd. AnotherMN-related protein was present in the tissue extract from a mammarypapilloma that protein was seen as a single band at about 48 kd (laneJ).

Clearly negative were the extracts from full-term placenta (lane B),normal mammary gland (lane K), hyperplastic endometrium (lane L), normalovaries (lane H), and from uterine myoma (lane I). Only extremelyslightly MN-related bands were seen in extracts from trophoblasts (lanesF and G) and from melanoma (lane P).

The observations that antigen related to p54/58N was expressed inclinical specimens of several types of human carcinomas but not ingeneral in normal tissues of the corresponding organs (exceptionsdelineated in Example 13) further strengthened the association of MNantigen with tumorigenesis. However, it should be noted that for humantumors, a normal tissue is never really an adequate control in thattumors are believed not to arise from mature, differentiated cells, butrather from some stem cells, capable of division and of differentiation.In body organs, such cells may be quite rare.

Example 7 MN Antigen in Animal Cell Lines

Since the MN gene is present in the chromosomal DNA of all vertebratespecies that were tested, MN-related antigen was searched for also incell lines derived from normal tissues and from tumors of several animalspecies. MN-related protein was found in two rat cell lines: one of themwas the XC cell line derived from rat rhabdomyosarcoma induced with Roussarcoma virus; the other was the Rat2-TK⁻ cell line. In extracts fromboth of those rat cell lines, a single protein band was found on theblots: its molecular weight on blots produced from a reducing gel andfrom a non-reducing gel was respectively 53.5 kd and 153 kd. FIG. 10shows the results with Rat2-Tk⁻ cell extracts (lane B), compared withextracts from MX-infected HeLa (lane A); the concentration of MN antigenin those two cell lines is very similar. The extracts were analyseddirectly (40 μl per lane).

MN-related protein from XC cells showed the same pattern as for Rat2-Tk⁻cells both under reducing and non-reducing conditions, except that itsconcentration was about 30× lower. The finding of a MN-relatedprotein—p53.5N—in two rat cell lines (FIGS. 10 and 12) provides thebasis for a model system.

None of the other animal cell lines tested contained detectable amountsof MN antigen, even when the highly sensitive immunoblot technique inwhich the MN antigens are concentrated was used. The MN-negative cellswere: Vero cells (African green monkey); mouse L cells; mouse NIH-3T3cells normal, infected with Moloney leukemia virus, or transformed withHarvey sarcoma virus; GR cells (mouse mammary tumor cells induced withMTV), and NMG cells (normal mouse mammary gland).

Example 8 Radioimmunoassays in Liquid Phase Using Recombinant MN Proteinfor MN-Specific Antibodies and for MN Antigen

The genetically engineered MN protein fused with glutathioneS-transferase—GEX-3X-MN—prepared and purified as described above waslabeled with ¹²⁵I by the chloramine T method [Hunter (1978)]. Thepurified protein enabled the development of a quantitative RIA forMN-specific antibodies as well as for MN antigens. All dilutions ofantibodies and of antigens were prepared in RIPA buffer (1% TRITON X-100and 0.1% sodium deoxycholate in PBS—phosphate buffered saline, pH 7.2),to which was added 1% of fetal calf serum (FCS). Tissue and cellextracts were prepared in RIPA buffer containing 1 mMphenylmethylsulfonylfluoride and 200 trypsin inhibiting units ofTrasylol (aprotinin) per ml, with no FCS. ¹²⁵I-labeled GEX-3X-MN protein(2.27 μCi/μg of TCA-precipitable protein) was before use diluted withRIPA+1% FCS, and non-specifically binding radioactivity was adsorbedwith a suspension of fixed protein A-Staphylococcus aureus cells (SAC).

In an RIA for MN-specific antibodies, MAb-containing ascites fluids ortest sera were mixed with ¹²⁵I-labeled protein and allowed to react in atotal volume of 1 ml for 2 hours at room temperature. Subsequently, 50μl of a 10% suspension of SAC [Kessler, supra] was added and the mixturewas incubated for 30 minutes. Finally, the SAC was pelleted, 3× washedwith RIPA, and the bound radioactivity was determined on a gammacounter.

Titration of antibodies to MN antigen is shown in FIG. 11. Ascitic fluidfrom a mouse carrying M75 hybridoma cells (A) is shown to have a 50%end-point at dilution 1:1.4×10⁻⁶. At the same time, ascitic fluids withMAbs specific for MX protein (M16 and M67) showed no precipitation of¹²⁵I-labeled GEX-3X-MN even at dilution 1:200 (result not shown). Normalrabbit serum (C) did not significantly precipitate the MN antigen;rabbit anti-MaTu serum (B), obtained after immunization with liveMX-infected HeLa cells, precipitated 7% of radioactive MN protein, whendiluted 1:200. The rabbit anti-MaTu serum is shown by immunoblot inExample 4 (above) to precipitate both MX and MN proteins.

Only one out of 180 human sera tested (90 control and 90 sera ofpatients with breast, ovarian or uterine cervical cancer) showed asignificant precipitation of the radioactively labeled MN recombinantprotein. That serum—L8—(D) was retested on immunoblot (as in Example 4),but it did not precipitate any p54/58N from MX-infected HeLa cells.Also, six other human sera, including KH (E), were negative onimmunoblot. Thus, the only positive human serum in the RIA, L8, wasreactive only with the genetically engineered product, but not withnative p54/58N expressed by HeLa cells.

In an RIA for MN antigen, the dilution of MAb M75, which in the previoustest precipitated 50% of maximum precipitable radioactivity (=dilution1:1.4×10⁻⁶) was mixed with dilutions of cell extracts and allowed toreact for 2 hours. Then, ¹²⁵I-labeled GEX-3X-MN (25×10³ cpm/tube) wasadded for another 2 hours. Finally, the radioactivity bound to MAb M75was precipitated with SAC and washed as above. One hundred percentprecipitation (=0 inhibition) was considered the maximum radioactivitybound by the dilution of MAb used. The concentration of the MN antigenin the tested cell extracts was calculated from an inhibition curveobtained with “cold” GEX-3X-MN, used as the standard (A in FIG. 12).

The reaction of radioactively labeled GEX-3X-MN protein with MAb M75enabled us to quantitate MN antigen directly in cell extracts. FIG. 12shows that 3′ ng of “cold” GEX-3X-MN (A) caused a 50% inhibition ofprecipitation of “hot” GEX-3X-MN; an equivalent amount of MN antigen ispresent in 3×10³ ng of proteins extracted from MaTu-infected HeLa (B) orfrom Rat2-Tk⁻ cells (C). Concentrations of MN protein in cell extracts,determined by this RIA, are presented in Table 1 below. It must beunderstood that the calculated values are not absolute, since MNantigens in cell extracts are of somewhat different sizes, and alsosince the genetically engineered MN protein is a product containingmolecules of varying size.

TABLE 1 Concentration of MN Protein in Cell Extracts Cells ng MN/mgtotal protein HeLa + MX 939.00 Rat2-Tk⁻ 1065.00 HeLa 27.50 XC 16.40 T241.18 HEF 0.00The data were calculated from the results shown in FIG. 12.

Example 9 Immunoelectron and Scanning Microscopy of Control and ofMX-Infected HeLa Cells

As indicated above in Example 1, MN antigen, detected by indirectimmunofluorescence with MAb M75, is located on the surface membranes andin the nuclei of MX-infected HeLa cells or in HeLa cells grown in densecultures. To elucidate more clearly the location of the MN antigen,immunoelectron microscopy was used wherein MAb M75 bound to MN antigenwas visualized with immunogold beads. [Herzog et al., “Colloidal goldlabeling for determining cell surface area,” IN: Colloidal Gold, Vol. 3(Hayat, M. A., ed.), pp. 139-149 (Academic Press Inc.; San Diego,Calif.).]

Ultrathin sections of control and of MX-infected HeLa cells are shown inFIG. 13 A-D. Those immuno-electron micrographs demonstrate the locationof MN antigen in the cells, and in addition, the strikingultrastructural differences between control and MX-infected HeLa. Acontrol HeLa cell (FIG. 13A) is shown to have on its surface very littleMN antigen, as visualised with gold beads. The cell surface is rathersmooth, with only two little protrusions. No mitochondria can be seen inthe cytoplasm. In contrast, MX-infected HeLa cells (FIGS. 13B and C)show the formation of abundant, dense filamentous protrusions from theirsurfaces. Most of the MN antigen is located on those filaments, whichare decorated with immunogold. The cytoplasm of MX-infected HeLacontains numerous mitochondria (FIG. 13C). FIG. 13D demonstrates thelocation of MN antigen in the nucleus: some of the MN antigen is innucleoplasm (possibly linked to chromatin), but a higher concentrationof the MN antigen is in the nucleoli. Again, the surface of normal HeLa(panels A and E of FIG. 13) is rather smooth whereas MX-infected HeLacells have on their surface, numerous filaments and “blebs”. Some of thefilaments appear to form bridges connecting them to adjacent cells.

It has been noted that in some instances of in vitro transformed cellscompared to their normal parent cells that one of the differences isthat the surface of normal cells was smooth whereas on the transformedcells were numerous hair-like protrusions [Darnell et al. “MolecularCell Biology,” (2nd edition) Sci. Am. Books; W.H. Freeman and Co., NewYork (1990)]. Under that criteria MX-infected HeLa cells, as seen inFIG. 13F, has a supertransformed appearance.

Further in some tumors, amplification of mitochondria has been described[Bernhard, W., “Handbook of Molecular Cytology,” pp. 687-715, Lima deFaria (ed.), North Holland Publishing Co.; Amsterdam-London (1972)].Such amplification was noted for MX-infected HeLa cells which stainedvery intensely with Janus' green, specific for mitochondria whereascontrol HeLa were only weakly stained.

It should be noted that electron microscopists were unable to find anystructural characteristics specific for tumor cells.

Example 10 Antisense ODNs Inhibit MN Gene Expression

To determine whether both of the p54/58N proteins were encoded by onegene, the following experiments with antisense ODNs were performed.Previously sparse-growing HeLa cells were seeded to obtain anovercrowded culture and incubated for 130 hours either in the absence orin the presence of two gene-specific ODNs complementary to the 5′ end ofMN mRNA. HeLa cells were subcultured at 8×10⁵ cells per ml of DMEM with10% FCS. Simultaneously, ODNs were added to the media as follows: (A)29-mer ODN1 (5′ CGCCCAGTGGGTCATCTTCCCCAGAAGAG 3′ [SEQ. ID. NO.: 3],complementary to positions 44-72 of FIG. 1A-1B) in 4 μM finalconcentration, (B) 19-mer ODN2 (5′ GGAATCCTCCTGCATCCGG 3′ [SEQ. ID. NO.:4], complementary to positions 12-30) in 4 μM final concentration and(C) both ODN1 and ODN2 in 2 μM final concentration each. (D) Cellstreated in the same way, but incubated without ODNs, served as acontrol. After 130 hours, extracts from the cells were prepared andanalyzed by immunoblotting using ¹²⁵I-labeled MAb M75. Protein extractsfrom the cells were analyzed by immunoblotting and RIA using MAb M75.FIG. 3 provides the immunoblot results of those experiments.

It was found that cultivation of HeLa cells with the ODNs resulted inconsiderable inhibition of p54/58N synthesis. The 19-mer ODN2 (FIG. 3B)in 4 μM final concentration was very effective; as determined by RIA, itcaused 40% inhibition, whereas the 29-mer ODN1 (4 μM) (FIG. 3A) and acombination of the two ODNs (FIG. 3C), each in 2 μM final concentration,were less effective in RIA showing a 25-35% decrease of the MN-relatedproteins. At the same time, the amount of different HeLa cell proteindetermined by RIA using specific MAb H460 was in all cell variantsapproximately the same. Most importantly was that on immunoblot it couldbe seen that specific inhibition by the ODNs affected both of thep54/58N proteins. Thus, we concluded that the MN gene we cloned codedfor both p54/58N proteins in HeLa cells.

The results indicated that the MN twin proteins arise by translation ofa single mRNA (consistent with the Northern blotting data). Thus, thetwin proteins may represent either differences in post-translationalmodification (phosphorylation, protease processing, etc.), or the use ofalternative translational initiation sites.

Example 11 Northern Blotting of MN mRNA in Tumorigenic andNon-Tumorigenic Cell Lines

FIG. 4 shows the results of Northern blotting of MN mRNA in human celllines. Total RNA was prepared from the following cell lines by theguanidinium thiocyanate-CsCl method: HeLa cells growing in a dense (A)and sparse (B) culture; CGL1 (H/F-N) hybrid cells (C); CGL3 (D) and CGL4(E) segregants (both H/F-T); and human embryo fibroblasts (F). Fifteenμg of RNA were separated on a 1.2% formaldehyde gel and blotted onto aHybond C Super membrane [Amersham]. MN cDNA NotI probe was labeled byrandom priming [Multiprime DNA labelling system; Amersham].Hybridization was carried out in the presence of 50% formamide at 42°C., and the final wash was in 0.1% SSPE and 0.1% SDS at 65° C. An RNAladder (0.24-9.5 kb) [BRL; Bethesda, Md. (USA)] was used as a sizestandard. Membranes were exposed to films at −70° C., with intensifyingscreens.

Detected was a 1.5 kb MN-specific mRNA only in two tumorigenic segregantclones—CGL3 and CGL4 (H/F-T), but not in the non-tumorigenic hybridclone CGL1 (H/F-N) or in normal human fibroblasts. Further, the 1.5 kbmRNA was found in the HeLa cells growing in dense (FIG. 4A) but not insparse (FIG. 4B) culture.

Thus, the results of the Northern blotting were consistent with otherexamples in regard to MN-related proteins being associated withtumorigenicity.

Example 12 Southern Blotting of Genomic DNAs from Different VertebrateSpecies to Detect MN Gene and Restriction Analysis of Genomic DNA ofHeLa Cells

FIG. 5 illustrates the detection of MN genes in the genomic DNAs ofvarious vertebrates by Southern blotting. Chromosomal DNA digested byPstI was as follows: (A) chicken; (B) bat; (C) rat; (D) mouse; (E)feline; (F) pig; (G) sheep; (H) bovine; (I) monkey; and (J) human HeLacells. Restriction fragments were separated on a 0.7% agarose gel andalkali blotted onto a Hybond N membrane [Amersham]. The MN cDNA probelabelling and hybridization procedures were the same as for the Northernblotting analyses shown in FIG. 4 and described in Example 11. TheSouthern blot of FIG. 5 made with PstI indicates that the MN gene isconserved in a single copy in all vertebrate genomes tested.

HeLa. Further, genomic DNA from HeLa cells was prepared as described byAusubel et al., Short Protocols in Molecular Biology [Greene PublishingAssociates and Wiley-Interscience; New York (1989)], digested withdifferent restriction enzymes, resolved on an agarose gel andtransferred to Hybond N+ membrane [Amersham]. The HeLa genomic DNA wascleaved with the following restriction enzymes with the results shown inFIG. 17 (wherein the numbers in parentheses after the enzymes indicatethe respective lanes in FIG. 17): EcoRI (1), EcoRV (2), HindIII (3),KpnI (4), NcoI (5), PstI (6), and PvuII (7), and then analyzed bySouthern hybridization under stringent conditions using MN cDNA as aprobe.

The prehybridization and hybridization using an MN cDNA probe labelledwith ³²P-dCTP by random priming [Multi-prime DNA labelling system;Amersham] as well as wash steps were carried out according to Amersham'sprotocols at high stringency. A 1 kb DNA Ladder [from BRL; Bethesda, Md.(USA)] was used as a size standard. Membranes were exposed to films at−70° C., with intensifying screens.

The Southern blotting analysis of HeLa chromosomal DNA showed that thegene coding for MN is present in the human genome in a single copy (FIG.17). The sizes and distribution of MN-positive restriction fragmentsobtained using the restriction enzymes KpnI, NcoI and HindIII indicatethat the MN gene contains introns, since those enzymes cut the MNgenomic sequences despite the absence of their restriction sites in MNcDNA.

Example 13 Immunohistochemical Staining of Tissue Specimens

To study and evaluate the tissue distribution range and expression of MNproteins, the monoclonal antibody M75 was used to stainimmunohistochemically a variety of human tissue specimens. The primaryantibody used in these immunohistochemical staining experiments was theM75 monoclonal antibody. A biotinylated second antibody andstreptavidin-peroxidase were used to detect the M75 reactivity insections of formalin-fixed, paraffin-embedded tissue samples. Acommercially available amplification kit, specifically the DAKO LSAB™kit [DAKO Corp., Carpinteria, Calif. (USA)] which provides matched,ready made blocking reagent, secondary antibody andstreptavidin-horseradish peroxidase was used in these experiments.

M75 immunoreactivity was tested according to the methods of thisinvention in multiple-tissue sections of breast, colon, cervical, lungand normal tissues. Such multiple-tissue sections were cut from paraffinblocks of tissues called “sausages” that were purchased from the City ofHope [Duarte, Calif. (USA)]. Combined in such a multiple-tissue sectionwere normal, benign and malignant specimens of a given tissue; forexample, about a score of tissue samples of breast cancers fromdifferent patients, a similar number of benign breast tissue samples,and normal breast tissue samples would be combined in one suchmultiple-breast-tissue section. The normal multiple-tissue sectionscontained only normal tissues from various organs, for example, liver,spleen, lung, kidney, adrenal gland, brain, prostate, pancreas, thyroid,ovary, and testis.

Also screened for MN gene expression were multiple individual specimensfrom cervical cancers, bladder cancers, renal cell cancers, and head andneck cancers. Such specimens were obtained from U.C. Davis MedicalCenter in Sacramento, Calif. and from Dr. Shu Y. Liao [Department ofPathology; St. Joseph Hospital; Orange, Calif. (USA)].

Controls used in these experiments were the cell lines CGL3 (H/F-Thybrid cells) and CGL1 (H/F-N hybrid cells) which are known to stainrespectively, positively and negatively with the M75 monoclonalantibody. The M75 monoclonal antibody was diluted to a 1:5000 dilutionwherein the diluent was either PBS [0.05 M phosphate buffered saline(0.15 M NaCl), pH 7.2-7.4] or PBS containing 1% protease-free BSA as aprotein stabilizer.

Immunohistochemical Staining Protocol

The immunohistochemical staining protocol was followed according to themanufacturer's instructions for the DAKO LSAB™ kit. In brief, thesections were dewaxed, rehydrated and blocked to remove non-specificreactivity as well as endogenous peroxidase activity. Each section wasthen incubated with dilutions of the M75 monoclonal antibody. After theunbound M75 was removed by rinsing the section, the section wassequentially reacted with a biotinylated antimouse IgG antibody andstreptavidin conjugated to horseradish peroxidase; a rinsing step wasincluded between those two reactions and after the second reaction.Following the last rinse, the antibody-enzyme complexes were detected byreaction with an insoluble chromogen (diaminobenzidine) and hydrogenperoxide. A positive result was indicated by the formation of aninsoluble reddish brown precipitate at the site of the primary antibodyreaction. The sections were then rinsed, counterstained withhematoxylin, dehydrated and cover slipped. Then the sections wereexamined using standard light microscopy. The following is an outline ofexemplary steps of the immunohistochemical staining protocol.

1. Series of ETOH-baths 100, 100, 95, 2 min. ± 1 min. each 95, 70% 2.dH₂0 wash - 2x 2 min. ± 1 min. each 3. 3% H₂0₂ as endogenous peroxidaseblock 5 min. 4. PBS wash - 2x 2 min. ± 1 min. 5. normal serum block(1.5% NGS) 30 min. 6. primary antibody (Mab M75) 60 min. ± 5 min. 7. PBSwash - 2x 2 min. ± 1 min. 8. biotinylated secondary antibody 20-30 min.± 2 min. 9. PBS wash - 2x 2 min. ± 1 min. 10. streptavidin-peroxidasereagent 20-30 min. ± 2 min. 11. PBS wash - 2x 2 min. ± 1 min. 12. DAB(150 ml Tris, 90 μl H₂0₂, 3 ml KPL 5-6 min. DAB) 13. PBS rinse, dH₂0wash 1-2 min. 14. Hematoxylin counterstain 2 min. ± 1 min. 15. wash withrunning tap water until clear 16. 0.05% ammonium hydroxide 20 sec. ± 10sec. 17. dH₂0 wash - 2x 3 min. ± 1 min. 18. dehydrate 70, 95, 95, 100,100% EtOH 2 min. ± 1 min. each 19. xylene 3x 3 min. ± 1 min. each 20.coverslip with Permount ™ [Fisher Scientific Pittsburgh, PA (USA)] 21.wait 10 min. before viewing results.

Interpretation. A deposit of a reddish brown precipitate over the plasmamembrane was taken as evidence that the M75 antibody had bound to a MNantigen in the tissue. The known positive control (CGL3) had to bestained to validate the assay. Section thickness was taken intoconsideration to compare staining intensities, as thicker sectionsproduce greater staining intensity independently of other assayparameters.

The above-described protocol was optimized for formalin-fixed tissues,but can be used to stain tissues prepared with other fixatives.

Results

Preliminary examination of cervical specimens showed that 62 of 68squamous cell carcinoma specimens (91.2%) stained positively with M75.Additionally; 2 of 6 adenocarcinomas and 2 of 2 adenosquamous cancers ofthe cervix also stained positively. In early studies, 55.6% (10 of 18)of cervical dysplasias stained positively. A total of 9 specimensincluding both cervical dysplasias and tumors, exhibited some MNexpression in normal appearing areas of the endocervical glandularepithelium, usually at the basal layer. In some specimens, whereasmorphologically normal-looking areas showed expression of MN antigen,areas exhibiting dysplasia and/or malignancy did not show MN expression.

M75 positive immunoreactivity was most often localized to the plasmamembrane of cells, with the most apparent stain being present at thejunctions between adjacent cells. Cytoplasmic staining was also evidentin some cells; however, plasma membrane staining was most often used asthe main criterion of positivity.

M75 positive cells tended to be near areas showing keratindifferentiation in cervical specimens. In some specimens, positivestaining cells were located in the center of nests of non-stainingcells. Often, there was very little, if any, obvious morphologicaldifference between staining cells and non-staining cells. In somespecimens, the positive staining cells were associated with adjacentareas of necrosis.

In most of the squamous cell carcinomas of the cervix, the M75immunoreactivity was focal in distribution, i.e., only certain areas ofthe specimen stained. Although the distribution of positive reactivitywithin a given specimen was rather sporadic, the intensity of thereactivity was usually very strong. In most of the adenocarcinomas ofthe cervix, the staining pattern was more homogeneous, with the majorityof the specimen staining positively.

Among the normal tissue samples, intense, positive and specific M75immunoreactivity was observed only in normal stomach tissues, withdiminishing reactivity in the small intestine, appendix and colon. Noother normal tissue stained extensively positively for M75.Occasionally, however, foci of intensely staining cells were observed innormal intestine samples (usually at the base of the crypts) or weresometimes seen in morphologically normal appearing areas of theepithelium of cervical specimens exhibiting dysplasia and/or malignancy.In such, normal appearing areas of cervical specimens, positive stainingwas seen in focal areas of the basal layer of the ectocervicalepithelium or in the basal layer of endocervical glandular epithelium.In one normal specimen of human skin, cytoplasmic MN staining wasobserved in the basal layer. The basal layers of these epithelia areusually areas of proliferation, suggesting the MN expression may beinvolved in cellular growth. In a few cervical biopsied specimens, MNpositivity was observed in the morphologically normal appearingstratified squamous epithelium, sometimes associated with cellsundergoing koilocytic changes.

Some colon adenomas (4 of 11) and adenocarcinomas (9 of 15) werepositively stained. One normal colon specimen was positive at the baseof the crypts. Of 15 colon cancer specimens, 4 adenocarcinomas and 5metastatic lesions were MN positive. Fewer malignant breast cancers (3of 25) and ovarian cancer specimens (3 of 15) were positively stained.Of 4 head and neck cancers, 3 stained very intensely with M75.

Although normal stomach tissue was routinely positive, 4 adenocarcinomasof the stomach were MN negative. Of 3 bladder cancer specimens (1adenocarcinoma, 1 non-papillary transitional cell carcinoma, and 1squamous cell carcinoma), only the squamous cell carcinoma was MNpositive. Approximately 40% (12 of 30) of lung cancer specimens werepositive; 2 of 4 undifferentiated carcinomas; 3 of 8 adenocarcinomas; 2of 8 oat cell carcinomas; and, 5 of 10 squamous cell carcinomas. Onehundred percent (4 of 4) of the renal cell carcinomas were MN positive.

In summary, MN antigen, as detected by M75 and immunohistochemistry inthe experiments described above, was shown to be prevalent in tumorcells, most notably in tissues of cervical cancers. MN antigen was alsofound in some cells of normal tissues, and sometimes in morphologicallynormal appearing areas of specimen exhibiting dysplasia and/ormalignancy. However, MN is not usually extensively expressed in mostnormal tissues, except for stomach tissues where it is extensivelyexpressed and in the tissues of the lower gastrointestinal tract whereit is less extensively expressed. MN expression is most often localizedto the cellular plasma membrane of tumor cells and may play a role inintercellular communication or cell adhesion. Representative results ofexperiments performed as described above are tabulated in Table 2.

TABLE 2 Immunoreactivity of M75 in Various Tissues POS/NEG TISSUE TYPE(#pos/#tested) liver, spleen, lung, normal NEG (all) kidney, adrenalgland, brain, prostate, pancreas, thyroid, ovary, testis skin normal POS(in basal layer) (1/1) stomach normal POS small intestine normal POScolon normal POS breast normal NEG (0/10) cervix normal NEG (0/2) breastbenign NEG (0/17) colon benign POS (4/11) cervix benign POS (10/18)breast malignant POS (3/25) colon malignant POS (9/15) ovarian malignantPOS (3/15) lung malignant POS (12/30) bladder malignant POS (1/3) head &neck malignant POS (3/4) kidney malignant POS (4/4) stomach malignantNEG (0/4) cervix malignant POS (62/68)

The results recorded in this example indicate that the presence of MNproteins in a tissue sample from a patient may, in general, dependingupon the tissue involved, be a marker signaling that a pre-neoplastic orneoplastic process is occurring. Thus, one may conclude from theseresults that diagnostic/prognostic methods that detect MN antigen may beparticularly useful for screening patient samples for a number ofcancers which can thereby be detected at a pre-neoplastic stage or at anearly stage prior to obvious morphologic changes associated withdysplasia and/or malignancy being evident or being evident on awidespread basis.

Example 14 Vaccine Rat Model

As shown above in Example 7, in some rat tumors, for example, the XCtumor cell line (cells from a rat rhabdomyosarcoma), a rat MN protein,related to human MN, is expressed. Thus a model was afforded to studyantitumor immunity induced by experimental MN-based vaccines. Thefollowing representative experiments were performed.

Nine- to eleven-day-old Wistar rats from several families wererandomized, injected intraperitoneally with 0.1 ml of either control ratsera (the C group) or with rat serum against the MN fusion proteinGEX-3X-MN (the IM group). Simultaneously both groups were injectedsubcutaneously with 10⁶ XC tumor cells.

Four weeks later, the rats were sacrificed, and their tumors weighed.The results are shown in FIG. 14. Each point on the graph represents atumor from one rat. The difference between the two groups—C and IM—wassignificant by Mann-Whitney rank test (U=84, α<0.025). The resultsindicate that the IM group of baby rats developed tumors about one-halfthe size of the controls, and 5 of the 18 passively immunized ratsdeveloped no tumor at all, compared to 1 of 18 controls.

Example 15 Expression of Full-Length MN cDNA in NIH 3T3 Cells

The role of MN in the regulation of cell proliferation was studied byexpressing the full-length cDNA in NIH 3T3 cells. That cell line waschosen since it had been used successfully to demonstrate the phenotypiceffect of a number of proto-oncogenes [Weinberg, R. A., Cancer Res., 49:3713 (1989); Hunter, T., Cell, 64: 249 (1991)]. Also, NIH 3T3 cellsexpress no endogenous MN-related protein that is detectable by Mab M75.

The full length MN cDNA was obtained by ligation of the two cDNA clonesusing the unique BamHI site and subcloned from pBluescript intoKpnI-SacI sites of the expression vector pSG5C. pSG5C was kindlyprovided by Dr. Richard Kettman [Department of Molecular Biology,Faculty of Agricultural Sciences, B-5030 Gembloux, Belgium]. pSG5C wasderived from pSG5 [Stratagene] by inserting a polylinker consisting of asequence having several neighboring sites for the following restrictionenzymes: EcoRI, XhoI, KpnI, BamHI, SacI, 3 times TAG stop codon andBglII.

The recombinant pSG5C-MN plasmid was co-transfected in a 10:1 ratio (10μg:1 μg) with the pSV2neo plasmid [Southern and Berg, J. Mol. Appl.Genet., 1: 327 (1982)] which contains the neo gene as a selectionmarker. The co-transfection was carried out by calcium phosphateprecipitation method [Mammalian Transfection Kit; Stratagene] into NIH3T3 cells plated a day before at a density of 1×10⁵ per 60 mm dish. As acontrol, pSV2neo was co-transfected with empty pSG5C.

Transfected cells were cultured in DMEM medium supplemented with 10% FCSand 600 μml⁻¹ of G418 [Gibco BRL] for 14 days. The G418-resistant cellswere clonally selected, expanded and analysed for expression of thetransfected cDNA by Western blotting using iodinated Mab M75.

For an estimation of cell proliferation, the clonal cell lines wereplated in triplicates (2×10⁴ cells/well) in 24-well plates andcultivated in DMEM with 10% FCS and 1% FCS, respectively. The medium waschanged each day, and the cell number was counted using a hemacytometer.

To determine the DNA synthesis, the cells were plated in triplicate in96-well plate at a density of 10⁴/well in DMEM with 10% FCS and allowedto attach overnight. Then the cells were labeled with ³H-thymidine for24 hours, and the incorporated radioactivity was counted.

For the anchorage-independent growth assay, cells (2×10⁴) were suspendedin a 0.3% agar in DMEM containing 10% FCS and overlaid onto 0.5% agarmedium in 60 mm dish. Colonies grown in soft agar were counted two weeksafter plating.

Several clonal cell lines constitutively expressing both 54 and 58 kdforms of MN protein in levels comparable to those found in LCMV-infectedHeLa cells were obtained. Selected MN-positive clones and negativecontrol cells (mock-transfected with an empty pSG5C plasmid) weresubjected to further analyses directed to the characterization of theirphenotype and growth behavior.

The MN-expressing NIH 3T3 cells displayed spindle-shaped morphology, andincreased refractility; they were less adherent to the solid support andsmaller in size. The control (mock transfected cells) had a flatmorphology, similar to parental NIH 3T3 cells. In contrast to thecontrol cells that were aligned and formed a monolayer with an orderedpattern, the cells expressing MN lost the capacity for growth arrest andgrew chaotically on top of one another (FIG. 23 a-d). Correspondingly,the MN-expressing cells were able to reach significantly higher (morethan 2×) saturation densities (Table 3) and were less dependent ongrowth factors than the control cells (FIG. 23 g-h).

MN transfectants also showed faster doubling times (by 15%) and enhancedDNA synthesis (by 10%), as determined by the amount of [³H]-thymidineincorporated in comparison to control cells. Finally, NIH 3T3 cellsexpressing MN protein grew in soft agar. The diameter of colonies grownfor 14 days ranged from 0.1 to 0.5 mm (FIG. 23 f); however, the cloningefficiency of MN transfectants was rather low (2.4%). Although thatparameter of NIH 3T3 cells seems to be less affected by MN than byconventional oncogenes, all other data are consistent with the idea thatMN plays a role in cell growth control.

TABLE 3 Growth Properties of NIH 3T3 Cells Expressing MN ProteinTransfected pSG5C/ pSG5C-MN/ DNA pSV2neo pSV2neo Doubling time^(a) 27.9± 0.5  24.1 ± 1.3 (hours) Saturation density^(b) 4.9 ± 0.2 11.4 ± 0.4(cells × 10⁴/cm²) Cloning <0.01  2.4 ± 0.2 efficiency (%)^(c) ^(a)Forcalculation of the doubling time, the proliferation rate ofexponentially growing cells was used. ^(b)The saturation cell densitywas derived from the cell number 4 days after reaching confluency.^(c)Colonies greater than 0.1 mm in diameter were scored at day 14.Cloning efficiency was estimated as a percentage of colonies per numberof cells plated, with correction for cell viability.

Example 16

Acceleration of G1 Transit and Decrease in Mitomycin C SensitivityCaused by MN Protein

For the experiments described in this example, the stable MNtransfectants of NIH 3T3 cells generated as described in Example 15 wereused. Four selected MN-positive clones and four control mock-transfectedclones were either used individually or in pools.

Flow cytometric analyses of asynchronous cell populations. For theresults shown in FIG. 24( a), cells that had been grown in dense culturewere plated at 1×10⁶ cells per 60 mm dish. Four days later, the cellswere collected by trypsinization, washed, resuspended in PBS, fixed bydropwise addition of 70% ethanol and stained by propidium iodinesolution containing RNase. Analysis was performed by FACStar using DNAcell cycle analysis software. [Becton Dickinson; Franklin Lakes, N.J.(USA)].

For the analyses shown in FIG. 24( b) and (c), exponentially growingcells were plated at 5×10⁵ cells per 60 mm dish and analysed as above 2days later. Forward light scatter was used for the analysis of relativecell sizes. The data were evaluated using Kolmogorov-Smirnov test[Young, J. Histochem. Cytochem., 25: 935 (1977)]. D is the maximumdifference between summation curves derived from histograms. D/s(n) is avalue which indicates the similarity of the compared curves (it is closeto zero when curves are similar).

The flow cytometric analyses revealed that clonal populationsconstitutively expressing MN protein showed a decreased percentage ofcells in G1 phase and an increased percentage of cells in G2-M phases.Those differences were more striking in cell populations grownthroughout three passages in high density cultures [FIG. 24( a)], thanin exponentially growing subconfluent cells [FIG. 24( b)]. Thatobservation supports the idea that MN protein has the capacity toperturb contact inhibition.

Also observed was a decrease in the size of MN expressing cells seen inboth exponentially proliferating and high density cultures. It ispossible that the MN-mediated acceleration of G1 transit is related tothe above-noted shorter doubling time (by about 15%) of exponentiallyproliferating MN-expressing NIH 3T3 cells. Also, MN expressing cellsdisplayed substantially higher saturation density and lower serumrequirements than the control cells. Those facts suggest thatMN-transfected cells had the capacity to continue to proliferate despitespace limitations and diminished levels of serum growth factors, whereasthe control cells were arrested in G1 phase.

Limiting conditions. The proliferation of MN-expressing and controlcells was studied both in optimal and limiting conditions. Cells wereplated at 2×10⁴ per well of 24-well plate in DMEM with 10% FCS. Themedium was changed at daily intervals until day 4 when confluency wasreached, and the medium was no longer renewed. Viable cells were countedin a hemacytometer at appropriate times using trypan blue dye exclusion.The numbers of cells were plotted versus time wherein each plot pointrepresents a mean value of triplicate determination.

The results showed that the proliferation of MN expressing and controlcells was similar during the first phase when the medium was reneweddaily, but that a big difference in the number of viable cells occurredafter the medium was not renewed. More than half of the control cellswere not able to withstand the unfavorable growth conditions. Incontrast, the MN-expressing cells continued to proliferate even whenexposed to increasing competition for nutrients and serum growthfactors.

Those results were supported also by flow cytometric analysis of serumstarved cells grown for two days in medium containing 1% FCS. While 83%of control cells accumulated in G0-G1 phase (S=5%, G2−M=12%), expressionof MN protein partially reversed the delay in G1 as indicated by cellcycle distribution of MN transfectants (G0−G1=65%, S=10%, G2−M=26%). Theresults of the above-described experiments suggest that MN protein mightfunction to release the G1/S checkpoint and allow cells to proliferateunder unfavorable conditions.

MMC. To test that assumption, unfavorable conditions were simulated bytreating cells with the DNA damaging drug mitomycin C (MMC) and thenfollowing their proliferation and viability. The mechanism of action ofMMC is thought to result from its intracellular activation andsubsequent DNA alkylation and crosslinking [Yier and Szybalski, Science,145: 55 (1964)]. Normally, cells respond to DNA damage by arrest oftheir cell cycle progression to repair defects and prevent acquisitionof genomic instability. Large damage is accompanied by markedcytotoxicity. However, many studies [for example, Peters et al., Int. J.Cancer, 54: 450 (1993)] concern the emergence of drug resistant cellsboth in tumor cell populations and after the introduction of oncogenesinto nontransformed cell lines.

The response of MN-transfected NIH 3T3 cells to increasingconcentrations of MMC was determined by continuous [³H]-thymidinelabeling. Cells were plated in 96-well microtiter plate concentration of10⁴ per well and incubated overnight in DMEM with 10% FCS to attach.Then the growth medium was replaced with 100 μl of medium containingincreasing concentrations of MMC from 1 μl/ml to 32 μg/ml. All the drugconcentrations were tested in three replicate wells. After 5 hours oftreatment, the MMC was removed, cells were washed with PBS and freshgrowth medium without the drug was added. After overnight recovery, thefractions of cells that were actively participating in proliferation wasdetermined by continuous 24-hr labeling with [³H]-thymidine. Theincorporation by the treated cells was compared to that of the control,untreated cells, and the proliferating fractions were considered as apercentage of the control's incorporation.

The viability of the treated cells was estimated three days later by aCellTiter 96 AQ Non-Radioactive Cell Proliferation Assay [Promega] whichis based on the bioreduction of methotrexate (MTX) into a water solubleformazan that absorbs light at 490 nm. The percentage of surviving cellswas derived from the values of absorbance obtained after subtraction ofbackground.

The control and MN-expressing NIH 3T3 cells showed remarkabledifferences in their responses to MMC. The sensitivity of theMN-transfected cells appeared considerably lower than the control's inboth sections of the above-described experiments. The results suggestedthat the MN-transfected cells were able to override the negative growthsignal mediated by MMC.

ATCC Deposits. The material listed below was deposited with the AmericanType Culture Collection (ATCC) at 10801 university Blvd., Manassas, Va.20110-2209 (USA). The deposits were made under the provisions of theBudapest Treaty on the International Recognition of DepositedMicroorganisms for the Purposes of Patent Procedure and Regulationsthereunder (Budapest Treaty). Maintenance of a viable culture is assuredfor thirty years from the date of deposit. The organism will be madeavailable by the ATCC under the terms of the Budapest Treaty, andsubject to an agreement between the Applicants and the ATCC whichassures unrestricted availability of the deposited hybridomas to thepublic upon the granting of patent from the instant application.Availability of the deposited strain is not to be construed as a licenseto practice the invention in contravention of the rights granted underthe authority of any Government in accordance with its patent laws.

Hybridoma Deposit Date ATCC # VU-M75 Sep. 17, 1992 HB 11128 MN 12.2.2Jun. 9, 1994 HB 11647

The description of the foregoing embodiments of the invention have beenpresented for purposes of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teachings. The embodiments were chosen anddescribed in order to explain the principles of the invention and itspractical application to enable thereby others skilled in the art toutilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated.

All references cited herein are hereby incorporated by reference.

1. A method of detecting or detecting and quantifying in a vertebrate sample MN protein or MN polypeptide comprising the steps of: (a) contacting said sample with at least one polyclonal antibody or fragment thereof which specifically binds to MN protein or to MN polypeptide; and (b) detecting or detecting and quantifying binding of said antibody or said antibody fragment in said sample; wherein said MN protein or MN polypeptide is encoded by a nucleotide sequence selected from the group consisting of: (1) SEQ ID NO: 5; (2) nucleotide sequences that hybridize under stringent hybridization conditions of 50% formamide at 42° C. with a final wash of 0.1% SSPE and 0.1% SDS at 65° C. to complement of SEQ ID NO: 5′s coding region; and (3) polynucleotides that differ from SEQ ID NO: 5 or from the polynucleotide sequences of (b) due to the degeneracy of the genetic code.
 2. The method according to claim 1 wherein said detecting or detecting and quantifying is by immunohistochemical staining.
 3. The method according to claim 1 which is diagnostic, or diagnostic and prognostic for neoplastic or preneoplastic and neoplastic disease, wherein said disease is associated with abnormal expression of MN protein.
 4. The method according to claim 1 wherein said polyclonal antibody is labeled.
 5. The method according to claim 1 wherein said method is in a format that is selected from the group consisting of Western blots, enzyme-linked immunosorbent assays, radioimmunoassays, competition immunoassays, dual antibody sandwich assays, immunohistochemical staining assays, agglutination assays, and fluorescent immunoassays.
 6. The method according to claim 1 wherein said polyclonal antibody specifically binds to one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 10-16.
 7. The method according to claim 6 wherein said group consists of SEQ ID NOS: 10, 11 and
 12. 8. The method according to claim 6 wherein said group consists of SEQ ID NOS: 10 and
 11. 9. The method according to claim 1 wherein the polyclonal antibody was prepared against recombinantly prepared MN protein or against recombinantly prepared MN fusion protein.
 10. The method according to claim 1, wherein one or more monoclonal antibodies that specifically bind to MN protein or MN polypeptide is or are used additionally in said detecting or detecting and quantifying method step (b).
 11. The method according to claim 10 wherein said one or more monoclonal antibodies are selected from the group consisting of the monoclonal antibodies designated M75 which are secreted by the hybridoma VU-M75 and the monoclonal antibodies designated MN12 that are secreted by the hybridoma MN 12.2.2, wherein said hybridomas have been deposited at the American Type Culture Collection (ATCC) under the ATCC Nos. HB 11128 and HB 11647, respectively.
 12. The method according to claim 10 wherein said one or more monoclonal antibodies are antigen binding antibody fragments comprising an MN protein binding region.
 13. The method according to claim 10 wherein said one or more monoclonal antibodies are genetically engineered antigen binding antibody fragments.
 14. The method according to claim 13 wherein said antigen binding antibody fragments are genetically engineered from the variable regions of the light and/or heavy chains (V_(H) and V_(L)).
 15. The method according to claim 10 wherein at least one of said one or more monoclonal antibodies is labeled.
 16. The method according to claim 1 wherein said MN protein or MN polypeptide has an amino acid sequence from SEQ ID NO:
 2. 17. The method according to claim 1 wherein said sample is obtained from a mammal.
 18. The method according to claim 1 wherein said sample is obtained from a human.
 19. The method according to claim 1 wherein said sample is selected from the group consisting of tissue specimens, tissue extracts and cell extracts.
 20. The method according to claim 19 wherein said tissue specimens are fresh, frozen or fixed.
 21. The method according to claim 19 wherein said tissue specimens are selected from the group consisting of cell smears, imprint preparations, histological sections from biopsied tissues, and histological sections from biopsied organs.
 22. The method according to claim 3 which is diagnostic or diagnostic and prognostic for neoplastic or preneoplastic and neoplastic diseases, wherein said diseases are associated with abnormal expression of MN protein, selected from the group consisting of mammary, urinary tract, ovarian, uterine, cervical, endometrial, vaginal, vulval, prostate, liver, lung, skin, thyroid, head and neck, gastrointestinal, and mesodermal pre-neoplastic and neoplastic diseases.
 23. A method of imaging neoplastic or preneoplastic and neoplastic disease in a patient comprising: (a) injecting said patient with polyclonal antibody which specifically binds to MN protein, and (b) detecting the binding of said antibody; wherein said MN protein is encoded by a nucleotide sequence selected from the group consisting of: (1) SEQ ID NO: 5; (2) nucleotide sequences that hybridize under stringent hybridization conditions of 50% formamide at 42° C. with a final wash of 0.1% SSPE and 0.1% SDS at 65° C. to complement of SEQ ID NO: 5′s coding region; and (3) polynucleotides that differ from SEQ ID NO: 5 or from the polynucleotide sequences of (b) due to the degeneracy of the genetic code. 